Methods using modified vaccinia virus

ABSTRACT

What are disclosed are methods for modifying the genome of vaccinia virus to produce vaccinia mutants, particularly by the introduction into the vaccinia genome of exogenous DNA; modified vaccinia prepared by such methods; certain DNA sequences and unmodified and genetically modified microorganisms involved as intermediates in such methods; and methods for infecting cells and host animals with such vaccinia mutants to provoke the amplification of exogenous DNA and proteins encoded by the exogenous DNA, including antigenic proteins, by said cells and host animals.

The invention described herein was made with the support of the FederalGovernment and the Federal Government has certain rights in theinvention.

This application is a division of application Ser. No. 08/306,259, filedSep. 13, 1994, now U.S. Pat. No. 5,583,028 which in turn is a divisionalof U.S. Ser. No. 08/228,926, filed Apr. 18, 1994 which in turn is acontinuation of application Ser. No. 07/881,995, filed May 4, 1992,abandoned, which in turn is a divisional of application Ser. No.537,882, filed Jun. 14, 1990, now U.S. Pat. No. 5,110,587, which is acontinuation of application Ser. No. 90,209, filed Aug. 27, 1987,abandoned which in turn is a divisional of 06/622,135, filed Jun. 19,1984, now U.S. Pat. No. 4,722,848 which in turn is acontinuation-in-part of application Ser. No. 446,824, filed, Dec. 8,1982, now U.S. Pat. No. 4,603,112, which in turn is acontinuation-in-part of application Ser. No. 334,456, filed Dec. 24,1981, now U.S. Pat. No. 4,769,330.

The present invention relates to modified vaccinia virus, to methods ofmaking and using the same, and to other modified and unmodifiedmicroorganisms, and to certain DNA sequences, produced or involved asintermediates in the production of modified vaccinia virus. More inparticular, the invention relates to vaccinia virus in which thenaturally occurring genome of the virus has been altered ("vacciniamutants") and to methods of making and using such vaccinia mutants, aswell as to other unmodified and genetically modified microorganisms, andto certain DNA sequences, produced or involved as intermediates in theproduction of vaccinia mutants.

Vaccinia virus is the prototypic virus of the pox virus family and, likeother members of the pox virus group, is distinguished by its large sizeand complexity. The DNA of vaccinia virus is similarly large andcomplex. Vaccinia DNA is about 120 megadaltons in size, for instance,compared with a DNA size of only 3.6 megadaltons for simian virus 40(SV40). The DNA molecule of vaccinia is double-stranded and terminallycrosslinked so that a single stranded circle is formed upon denaturationof the DNA. Vaccinia DNA has been physically mapped using a number ofdifferent restriction enzymes and a number of such maps are presented inan article by Panicali et al., J. Virol. 37, 1000-1010 (1981) whichreports the existence of two major DNA variants of the WR strain ofvaccinia virus (ATCC No. VR 119), which strain has been most widely usedfor the investigation and characterization of pox viruses. The twovariants differ in that the S("small") variant (ATCC No. VR 2034) has a6.3 megadalton deletion not occurring in the DNA of the L("large")variant (ATCC No. VR 2035). Maps obtained by treatment of the variantswith the restriction enzymes Hind III, Ava I, Xho I, Sst I, and Sma Iare presented in the aforementioned article.

Vaccinia, a eukaryotic virus, reproduces entirely within the cytoplasmof a host cell. It is a lytic virus, i.e. a virus, the replication ofwhich in a cell results in lysis of the cell. The virus is considerednon-oncogenic. The virus has been used for approximately 200 years invaccines for inoculation against smallpox and the medical profession iswell acquainted with the properties of the virus when used in a vaccine.Although inoculation with vaccinia is not without risk, the risks are onthe whole well known and well defined and the virus is consideredrelatively benign.

At the heart of the present invention is the modification of thenaturally occurring vaccinia genome to produce vaccinia mutants byrearrangement of the natural genome, by the removal of DNA from thegenome, and/or by the introduction into the naturally occurring vacciniagenome of DNA which disrupts the naturally occurring genome ("foreignDNA"). Such foreign DNA may be naturally occurring in vaccinia or may besynthetic or may be naturally occurring in an organism other thanvaccinia. If genetic information is present in this foreign DNA, thepotential exists for the introduction of this information into aeukaryote via modified vaccinia virus. That is, the modified virusrepresents a relatively innocuous eukaryotic cloning vector from whichgenetic information has been deleted, or into which information has beeninserted, or in which genetic information has been rearranged. Since thevirus replicates within the cytoplasm of an infected cell, modifiedvaccinia virus represents a unique eukaryotic cloning vector unlike anyother so far considered or currently under investigation.

This discovery has a number of useful consequences, among which are (A)novel methods for vaccinating mammals susceptible to vaccinia to inducein them an antibody response to antigens coded for by foreign DNAinserted into the vaccinia virus, (B) novel methods for the productionby eukaryotic cells of biological products other than antigens, and (C)novel methods for the introduction into human or animal individuals orpopulations of missing genes or of genetic material for themodification, replacement, or repair of defective genes in theindividuals or populations.

Suitably modified vaccinia mutants carrying exogenous genes which areexpressed in a host as an antigenic determinant eliciting the productionby the host of antibodies to the antigen represent novel vaccines whichavoid the drawbacks of conventional vaccines employing killed orattenuated live organisms. Thus, for instance, the production ofvaccines from killed organisms requires the growth of large quantitiesof the organisms followed by a treatment which will selectively destroytheir infectivity without affecting their antigenicity. On the otherhand, vaccines containing attenuated live organisms always present thepossibility of a reversion of the attenuated organism to a pathogenicstate. In contrast, when a modified vaccinia mutant suitably modifiedwith a gene coding for an antigenic determinant of a disease-producingorganism is used as a vaccine, the possibility of reversion to apathogenic organism is avoided since the vaccinia virus contains onlythe gene coding for the antigenic determinant of the disease producingorganism and not those genetic portions of the organism responsible forthe replication of the pathogen.

The present invention offers advantages even with respect to newtechnology employing genetic engineering involving the production of anantigen by a recombinant prokaryotic organism containing a plasmidexpressing a foreign antigenic protein. For instance, such technologyrequires the production of large amounts of the recombinant prokaryoticcells and subsequent purification of the antigenic protein producedthereby. In contrast, a modified vaccinia virus used for innoculationaccording to the present invention replicates within the innoculatedindividual to be immunized, thereby amplifying the antigenic determinantin vivo.

A further advantage of the use of vaccinia mutants as vectors ineukaryotic cells as vaccines or for producing biological products otherthan antigens is the possibility for post-translational modifications ofproteins produced by the transcription of exogenous genes introducedinto the cell by the virus. Such post-translational modifications, forinstance glycosylation of proteins, are not likely in a prokaryoticsystem, but are possible in eukaryotic cells where additional enzymesnecessary for such modifications are available. A further advantage ofthe use of vaccinia mutants for inoculation is the possibility ofamplification of the antibody response by the incorporation, into themutant, of tandem repeats of the gene for the antigen or of additionalgenetic elements which stimulate the immune response, or by the use of astrong promoter in the modified virus. A similar advantage holds in theproduction of biological products other than antigens.

Returning to a more detailed consideration of the vaccinia genome, thecross-linked double strands of the DNA are characterized by invertedterminal repeats each approximately 8.6 megadaltons in length,representing about 10 kilobasepairs (kbp). Since the central portions ofthe DNA of all pox viruses are similar, while the terminal portions ofthe viruses differ more strongly, the responsibility of the centralportion for functions common to all the viruses, such as replication, issuggested, whereas the terminal portions appear responsible for othercharacteristics such as pathogenicity, host range, etc. If such a genomeis to be modified by the rearrangement or removal of DNA fragmentstherefrom or the introduction of exogenous DNA fragments thereinto,while producing a stable viable mutant, it is evident that the portionof the naturally-occurring DNA which is rearranged, removed, ordisrupted by the introduction of exogenous DNA thereinto must benon-essential to the viability and stability of the host, in this casethe vaccinia virus. Such non-essential portions of the genome have beenfound to be present in the WR strain of vaccinia virus, for instancewithin the region present within the L-variant but deleted from theS-variant or within the Hind III F-fragment of the genome.

The modification of vaccinia virus by the incorporation of exogenousgenetic information can be illustrated by the modification of the WRstrain of vaccinia virus in the Hind III F-fragment thereof toincorporate into that fragment a gene of herpes simplex virus type I(HSV) responsible for the production of thymidine kinase (TK). TK is anenzyme which phosphorylates the nucleoside thymidine to form thecorresponding mono-phosphorylated nucleotide which is subsequentlyincorporated into DNA.

The HSV TK gene represents DNA foreign to vaccinia virus which isconvenient to introduce into vaccinia according to the present inventionfor a number of reasons. First, the gene is relatively readily availablepresent in a herpes simplex virus DNA fragment that is produced bydigestion with Bam HI endonuclease, as reported by Colbere-Garapin etal. in Proc. Natl. Acad. Sci. USA 76, 3755-3759 (1979). Second, this HSVBam HI fragment has been introduced into plasmids and into eukaryoticsystems in the prior art, for instance as reported by Colbere-Garapin etal., loc. cit. and by Wigler et al., Cell 11, 223-232 (1977). Third,experience has shown that if HSV TK can be introduced as an exogenousgene into a eukaryotic system, and is expressed--which requiresunambiguous and faithful translation and transcription of the geneticlocus--, then other exogenous genes can similarly be introduced andexpressed.

    ______________________________________                                        ORGANISM               ATCC NO.                                               ______________________________________                                        WR strain of vaccinia virus                                                                          VR 119                                                   WR strain of vaccinia virus, VR 2034                                          S-variant                                                                     WR strain of vaccinia virus, VR 2035                                          L-variant                                                                     Syrian hamster kidney cells CCL-10                                            CV-1 cells CCL-70                                                             VP-1 VR 2032                                                                  VP-2 VR 2030                                                                  VP-3 VR 2036                                                                  VP-4 VR 2033                                                                  VP-5 VR 2028                                                                  VP-6 VR 2029                                                                  VP-7 VR 2042                                                                  VP-8 VR 2053                                                                  VP-9 VR 2043                                                                  VP-10 VR 2044                                                                 VP-11 VR 2045                                                                 VP-12 VR 2046                                                                 VP-13 VR 2047                                                                 VP-14 VR 2048                                                                 VP-16 VR 2050                                                                 VP-17 VR 2051                                                                 VP-18 VR 2052                                                                 VP-22 VR 2054                                                                 VTK-79 VR 2031                                                                VTK-79L VR 2056                                                               VP-53 VR 2060                                                                 VP-59 VR 2061                                                                 VP-60 VR 2062                                                                 VTK-11 VR 2027                                                                pDP122B 39736                                                                 pDP252 39735                                                                  PBL540A 68574                                                               ______________________________________                                    

better understanding of the present invention will be had by referringto the accompanying drawings, in which

FIG. 1 is a map of the aforementioned L- and S-variants of the WR strainof vaccinia determined using Hind III as a restriction enzyme andshowing the deletion of sequences in the terminal C fragment of theL-variant, which deletion is outside the terminal repeat section of thegenome. The deleted DNA sequences are unique to the L structure and,since the growth of the S- and L-variants is identical, this deletedregion must be non-essential;

FIG. 2 shows the vaccinia Hind III F-fragment in greater detail,including two further restriction sites therein, namely Sst I and BamHI, at least the latter of which sites offers a locus at which exogenousDNA can be introduced into the vaccinia Hind III F-fragment withoutdisturbing any essential vaccinia genes;

FIGS. 3A-C schematically show a method for the introduction of the HSVTK gene into the vaccinia Hind III F-fragment;

FIGS. 4A-C are a restriction map of certain vaccinia mutants producedaccording to the present invention and shows in detail the position ofthe HSV TK inserts present in the Hind III F-fragment in two such virusmutants, designated herein as VP-1 and VP-2;

FIG. 5 is a table summarizing certain techniques useful in screeningpossible recombinant viruses to determine the presence or absence of theHSV TK gene therein; and

FIGS. 6A-C are restriction maps of the left-hand terminal portion of thevaccinia WR genome showing the relationship of various restrictionfragments to the unique L-variant DNA sequence deleted from thecorresponding S-variant.

FIGS. 7A-C schematically show a method for constructing a new plasmid,pDP 120, which contains a portion of the vaccinia Hind III F-fragmentcombined with pBR 322, but which plasmid is of lower molecular weightthan plasmid pDP 3, shown in FIG. 3B above.

FIGS. 8A-D schematically show the construction of two plasmids, pDP 301Aand pDP 301B, which permit the incorporation of the DNA sequence of pBR322 into vaccinia virus to produce vaccinia mutants VP 7 and VP 8.

FIGS. 9A-C schematically show the construction of a virus mutant, VP 9,into the genome of which the influenza hemagglutinin gene (HA) has beenincorporated using a technique like that shown in FIGS. 8A-D.

FIGS. 10A-C schematically show the construction of a further vacciniamutant, VP 10, also containing the influenza hemagglutinin (HA) gene,but prepared directly by in vivo recombination using VP 7.

FIGS. 11A-E show the construction of two vaccinia mutants, designated VP11 and VP 12, each of which contains in its genome the DNA sequencecoding for the surface antigen of hepatitis B virus incorporatedthereinto by in vivo recombination of vaccinia virus VTK⁻ 79 with,respectively, newly constructed plasmids pDP 250B and pDP 250A.

FIGS. 12A-D schematically show the construction of a new plasmid, pDP252, which combines pBR 322 with a portion of the hepatitis B virus(HBV) genome, which portion is entirely within that region of the HBVregion genome which codes for the surface antigen. The Figures show theintroduction of the resulting pDP 252 plasmid into vaccinia mutant VP 8with the resultant formation of a further vaccinia variant identified asVP 13.

FIGS. 13A-C show the construction of two further plasmids, pBL 520 A andpBL 520 B, and their insertion into VP 7 to produce two further vacciniamutants, VP 16 and VP 14, each containing the DNA sequence of herpesvirus I which codes for the production of herpes glycoproteins gA+gB,two of the principal immunogenic proteins of herpes simplex virus typesI and II.

FIGS. 14A-C show the construction of two further plasmids, pBL 522 A and522 B, incorporating the 5.1 md Bam HI fragment G shown in FIG. 13A aspresent in the Eco RI herpes F-fragment [De Luca et al., Virology 122,411-423 (1982)]

FIGS. 15A-F show the construction of a further vaccinia variant, VP 22 ,in which foreign DNA, namely a herpes Bgl/Bam TK fragment, has beeninserted into the vaccinia genome in a non-essential portion other thanthe F-fragment thereof.

FIG. 16 shows the construction of a further plasmid, pRW 120.

FIG. 17 shows the construction of a further vaccinia variant, vP 53,expressing influenza virus hemagglutinin (HA).

FIG. 18 shows the construction of a further vaccinia variant, vP 59,expressing the hepatitis B virus surface antigen (HBsAg).

FIG. 19 shows the construction of yet another vaccinia variant, vP 60,expressing the herpes simplex virus glycoprotein D (HSVgD).

FIG. 20 is a plot of antibody response in rabbits inoculated withvaccinia variant vP 59 or with both vP 59 and vP 60.

Referring to FIG. 1, if the L- and S-variants of the vaccinia virus aresubjected to the action of Hind III, a restriction enzyme well known inthe prior art and commercially available, the virus genomes arerespectively cleaved into 15 or 14 segments designated with the lettersA through O, with the letter A used to designate the largest fragmentand the letter O used to designate the smallest. The electrophoreticseparation of the restriction fragments is described and shown in theaforementioned publication of Panicali et al., J. Virol. 37, 1000-1010(1981). The F-fragment obtained in this manner from either the L- orS-variants has a molecular weight of 8.6 megadaltons. The position ofthe F-fragment is shown on the restriction map presented as FIG. 1accompanying the application and a restriction map of the F-fragment isshown in FIG. 2. The restriction enzyme Hind III recognizes thenucleotide sequence -AAGCTT- and cleaves the DNA between the adjacentadenosine groups to give fragments having "sticky ends" with thesequence AGCT-. Since larger quantities of the Hind III F-fragment ofvaccinia than are readily obtainable by restriction of the vacciniagenome are required for manipulation according to the present invention,the F-fragment is inserted into a plasmid cloning vector for purposes ofamplification.

Namely, the vaccinia rind III F-fragment produced in this manner isconveniently introduced into the plasmid pBR 322 which is cut only onceby a number of restriction enzymes, including Hind III. The pBR 322plasmid was first described by Bolivar et al. in Gene 2, 95-113 (1977)and is now commercially available within the United States from a numberof sources.

The location of the Hind III cleavage site on the pBR 322 plasmid isindicated in FIG. 3A relative to cleavage sites of Eco RI and Bam HI,which are other restriction enzymes. If the pBR 322 plasmid is cut withHind III and the resultant cleaved DNA is mixed with vaccinia Hind IIIF-fragment, and if the fragments are ligated with T₄ DNA ligase, assuggested in FIG. 3A, the F-fragment is incorporated into the plasmid toproduce the novel plasmid pDP 3 shown schematically in FIG. 3B andhaving a molecular weight of approximately 11.3 megadaltons. Thevaccinia mind III F-fragment includes approximately 13 kilobasepairs incomparison with the 4.5 kilobasepairs found in the pBR 322 portion ofpDP 3. T₄ DNA ligase is a commercially available enzyme and theconditions for its use in the manner indicated are well known in theart.

The pDP 3 plasmid is now introduced into a microorganism such asEscherichia coli (E. coli) by transformation for purposes of replicatingthe Hind III F-fragment for recovery of larger quantities of theF-fragment. These techniques of cleaving a plasmid to produce linear DNAhaving ligatable termini and then inserting exogenous DNA havingcomplementary termini in order to produce a replicon (in this case thepBR 322 containing vaccinia Hind III F-fragment) are known in the art,as is the insertion of the replicon into a microorganism bytransformation (cf. U.S. Pat. No. 4,237,224).

Unmodified pBR 322 plasmid confers ampicillin resistance (Amp^(R)) andtetracycline resistance (Tet^(R)) to its host microorganism, in thiscase E. coli. However, since Hind III cuts the pBR 322 plasmid in theTet^(R) gene, the introduction of the vaccinia Hind III F-fragmentdestroys the Tet^(R) gene and tetracycline resistance is lost. Hence,the E. coli transformants containing the pDP 3 plasmid can bedistinguished from untransformed E. coli by the simultaneous presence ofresistance to ampicillin and susceptibility to tetracycline. It is theseE. coli transformed with pDP 3 which are grown in large quantities andfrom which large quantities of the pDP 3 are recovered.

The conditions under which plasmids can be amplified in E. coli are wellknown in the art, for example from the paper of Clewel, J. Bacteriol.110, 667-676 (1972). The techniques of isolating the amplified plasmidfrom the E. coli host are also well known in the art and are described,for instance, by Clewel et al. in Proc. Natl. Acad. Sci. USA 62,1159-1166 (1969).

In a similar fashion, the pBR 322 plasmid can be conveniently cleaved bytreatment with the restriction enzyme Bam HI and a modified plasmid canbe prepared by the insertion thereinto of a Bam HSV TK fragment, all asdiscussed in the aforementioned work of Colbere-Garapin et al., loc.cit. The modified plasmid containing the Bam HI fragment which includesthe HSV TK gene can again be introduced into E. coli by known methodsand the transformed bacteria grown for amplification of the plasmid inlarge quantities. The amplified Bam HSV TK-pBR 322 recombinant plasmidis subsequently cleaved with Bam HI to isolate the Bam HI fragmentcontaining the HSV TK gene using the same prior art techniques mentionedearlier with regard to the amplification of the Hind III F-fragment ofvaccinia.

To construct a recombinant plasmid having the Bam HI HSV TK fragmentincluded within the vaccinia Hind III F-fragment, the pDP 3 plasmid isnext subjected to a partial restriction with Bam HI such that only oneof the two Bam HI cleavage sites within the plasmid is cleaved, i.e.either that Bam HI site within the Hind III F-fragment or the Bam HIsite within the pBR 322 portion of the pDP 3 plasmid, as shown in FIG.3B. The cleaved, now-linear, DNA is then combined with purified Bam HSVTK fragment. The linear segments are combined and ligated by treatmentwith T₄ DNA ligase, again using techniques known in the art.

The combination of the Bam HSV TK fragment with the cleaved pDP 3plasmid is a random or statistical event leading to the possibleproduction of numerous species formed by various combinations of thefragments present in the mixture, all of which have identical "stickyends". Thus, one possibility is the simple rejoining of the Bam HIcleaved ends of the pDP 3 plasmid to reform the circular plasmid.Another possibility is the joinder of two or more Bam HSV TK fragmentsin either of two orientations. Further, the Bam HSV TK fragment (or amultiple thereof) may be combined with the linear DNA of a pDP 3 plasmidwhich has been cleaved at the Bam HI site within the pBR 322 portion,again in either of two orientations, or one or more Bam HSV TK fragmentsmay be combined, again in either of two orientations, with linear pDP 3DNA which has been cleaved at the Bam HI site within the vaccinia HindIII F-fragment portion of the pDP 3 plasmid.

To permit the identification and separation of these variouspossibilities, the products of ligation are inserted into a unicellularmicroorganism such as E. coli by techniques like those described earlierand known in the art. The E. coli thus treated are then grown on amedium containing ampicillin. Those bacteria which contain any plasmidare ampicillin resistant because all such plasmids contain that gene ofpBR 322 which confers ampicillin resistance. Hence, all survivingbacteria are transformants which are then screened further to determinethe presence or absence of the Bam HSV TK fragment possibly present.

To accomplish this, those bacteria containing any TK gene are identifiedby hybridization with radio-labelled TK DNA. If the TK gene is presentin the bacterium, the radio-labelled TK DNA will hybridize with thatportion of the plasmid present in the bacterium. Since the hybrid isradioactive the colonies containing TK within their plasmids can bedetermined by means of autoradiography. The bacteria containing TK canin turn be grown. Finally then, bacteria containing plasmids having theTK incorporated within the pBR 322 portion can be identified andseparated from those having the TK fragment in the vaccinia Hind IIIF-fragment by analysis with restriction endonucleases.

More in detail, the bacteria surviving growth on nutrient agar platescontaining ampicillin are partially transferred to a nitrocellulosefilter by contact of the filter with the plate. The bacteria remainingon the plate are regrown and the bacteria which have been transferred tothe nitrocellulose filter to create a replica of the original plate arenext treated to denature their DNA. Denaturation is effected, forexample, by treatment of the transferred bacteria with sodium hydroxide,followed by neutralization and washing. Subsequently, the now-denaturedDNA present on the nitrocellulose filter is hybridized by treatment withHSV Bam TK containing radioactive ³² P. The nitrocellulose filter sotreated is next exposed to X-ray film which darkens in those portions inwhich hybridization with the radio-labelled Bam HSV TK has taken place.The exposed darkened X-ray film is next compared with the original plateand those colonies growing on the original plate corresponding to thecolonies causing darkening of the X-ray film are identified as thosecontaining a plasmid in which Bam HSV TK is present.

Finally, to discriminate between those bacteria containing a plasmid inwhich the Bam HSV TK gene has been incorporated within the pBR 322portion of the plasmid from those wherein Bam HSV TK is present in theF-fragment of the plasmid, small cultures of the bacteria are grown andthe plasmids are isolated therefrom by a mini-lysis technique known inthe art and described in the paper of Holmes et al., Anal. Bioch. 114193-197 (1981). The plasmids are next digested with the restrictionenzyme Hind III which cleaves the circular plasmid at the two points oforiginal joinder of the F-fragment with the pBR 322 DNA chain. Themolecular weight of the digestion product is next determined byelectrophoresis on agarose gels, with the distance of migration in thegels being a measure of the molecular weight.

If the Bam HSV TK fragment or a multiple thereof is found in theF-segment of the digested plasmid, the gel will show the presence of thepBR 322 fragment plus a second fragment having a molecular weightgreater than that of the F-fragment by the molecular weight of the BamHSV TK DNA segment or segments included therein. Conversely, if the BamHSV TK is present in the pBR 322, electrophoresis will show the presenceof an F-fragment of the usual molecular weight plus a further fragmentlarger than pBR 322 by the molecular weight of the Bam HSV TK fragmentor fragments present therein. Those bacteria in which modification withBam HSV TK has occurred in the pBR 322 portion of the plasmid arediscarded: the remaining bacteria have been modified in the F-fragmentportion of the plasmid therein. It is these plasmids which are used forincorporation of the Bam HSV TK fragment into vaccinia.

As mentioned earlier, the combination of the DNA fragments to regeneratea plasmid is a random event according to which a number of whichdifferent plasmid structures having Bam HSV TK in the F-fragment canresult.

To determine the orientation of the Bam HSV TK fragment within theF-fragment, as well as the number of such Bam HSV TK fragments possiblypresent, the plasmids are recovered from each of those bacterialcolonies which are known to have an Bam HSV TK fragment present in theF-fragment of the plasmid. The mini-lysis technique mentioned earlierherein is used for this purpose. The plasmids are then again subjectedto restriction analysis, this time using the commercially availablerestriction enzyme Sst I. Since each Bam HSV TK fragment has an Sst Irestriction site therein, and since the F-fragment of vaccinia similarlyhas a single Sst I restriction site therein (cf. the representation ofthese fragments in FIGS. 3B and 3A respectively), different numbers offragments of differing molecular weights can be detected byelectrophoresis on agarose gels, the number and molecular weight of thesegments being dependent on the orientation of the Bam HSV TK fragmentwithin the F-fragment and the number of such Bam TK fragments present.Orientation of the Bam TK fragment within the F-fragment can be detectedbecause of the asymmetry of the Bam HSV TK fragment with respect to theSst I site therein (cf. FIG. 3B).

For instance, in the particular experiments under discussion, sixbacterial colonies each having one or more Bam HSV TK fragments presentin the F-fragment of the plasmid were found among the E. colitransformants. After restriction analysis of the plasmids in thesebacteria along the lines discussed above, two of the recombinantplasmids were chosen for further study because the direction oforientation of the Bam HSV TK fragment within the F-fragment was inopposite directions.

At this point, the reader is reminded that the introduction of the HSVTK gene into the F-fragment of vaccinia, as discussed in detail above,is merely exemplary of one of many possible means of modifying thevaccinia genome to produce desirable vaccinia mutants. Thus, theintroduction of the same exogenous gene into another portion of thevaccinia genome, or the introduction of different genetic material intothe vaccinia F-fragment or into some other fragment, all may requiremodification of the exemplary scheme, discussed above, for theidentification of recombinant organisms.

For instance, digestion of the vaccinia L-variant within Ava I yields afragment, H, entirely with the region deleted from the S-variant (cf.FIG. 6A and the discussion thereof infra). This H-fragment contains BamHI sites permitting the introduction thereinto of the HSV TK gene. Thesame scheme for identifying F-Fragment-HSV TK recombinants can be usedfor identifying such H-fragment recombinants also.

Indeed, schemes for the construction and identification ofF-fragment-HSV TK recombinants, alternative to that disclosed in detailabove by way of illustration, do exist. For instance, the Bam HI site inpBR 322 can be removed by cleavage of the plasmid with Bam HI andtreatment with DNA polymerase I to "fill in" the "sticky ends". Thisproduct is then cut with Hind III and the linear fragment is treatedwith alkaline phosphatase to prevent recircularization of the plasmidupon ligation. However, foreign DNA, and particularly the vaccinia HindIII F-fragment, can be ligated to the treated pBR 322 and the resultingplasmid will recircularize. Now, treatment with Bam HI effects cleavageof the plasmid only within the vaccinia F-fragment portion thereof.Subsequent treatment of the cleavage product with alkaline phosphataseand ligation with the Bam HI HSV TK fragment will produce recombinantswith high efficiency so that the recombinants can be screened byrestriction endonuclease cleavage and gel electrophoresis. Thistechnique eliminates the time-consuming steps of discriminating betweenrecombinants having HSV TK in the pBR 322 portion or in the F-fragmentand colony hybridization.

Returning now to further discussion of the plasmids produced in theexemplary mutation of vaccinia by the introduction of HSV TK into thevaccinia F-fragment, the two recombinant plasmids chosen for furtherstudy are shown in FIG. 3C, where they are identified as a first novelplasmid, pDP 132, incorporating one Bam HSV TK fragment within thevaccinia Hind III F-portion, and a second novel plasmid, pDP 137, inwhich two Bam HSV TK fragments joined "head to tail" have beenincorporated. The single fragment of Bam HSV TK has been incorporatedwithin pDP 132 in the opposite sense in which two Bam TK fragments havebeen included in tandem in pDP 137. Namely, the region of the TK genewithin the Bam HI fragment which codes for the 5'-end of mRNA producedby the gene is located between the Sst I cleavage site and the nearer ofthe two Bam HI sites thereto (again cf. FIG. 3B). The direction oftranscription of the HSV TK gene on the Bam TK fragment proceeds fromthe 5'-end to the 3'-end and will be in a clockwise direction in pDP 132as shown in FIG. 3C. [cf. Smiley et al., Virology 102, 83-93 (1980)].Conversely, since the Bam TK fragments included in tandem in pDP 137have been incorporated in the reverse sense, transcription of the HSV TKgenes contained therein will be in the opposite direction, namely in acounter-clockwise direction. The direction of inclusion of the Bam HSVTK fragment within the vaccinia Hind III F-fragment may be of importancein case promotion of transcription of the HSV TK gene is initiated by apromoter site within the F-fragment itself. However, HSV promoter sitesdo exist within the Bam HSV TK fragment itself, so that transcription ofthe HSV TK gene may occur no matter in which direction the Bam HSV TKfragment and HSV TK gene have been incorporated within the vaccinia HindIII F-fragment.

Those E. coli transformants containing pDP 132 or pDP 137 are next grownto produce large amounts of the plasmids for further processing. When asufficient amount of the plasmid DNA has been isolated, restriction withHind III yields a modified vaccinia Hind III F-fragment having the HSVTK gene therein. This modified Hind III F-fragment is now introducedinto vaccinia virus by novel methods, described below in greater detail,in order to produce an infectious entity.

To review the prior art, at present the vector principally used forintroducing exogenous DNA into eukaryotic cells is SV40. The DNA of SV40is circular and can be treated much like a plasmid. That is, thecircular DNA is cleaved with a restriction enzyme, combined withexogenous DNA, and ligated. The modified DNA can be introduced intoeukaryotic cells, for instance animal cells, by standard techniques [cf.Hamer et al., Nature 281, 35-40 (1979)]. The DNA is infectious and willreplicate in the nucleus of the cell producing viable mutated viruses.In contrast, vaccinia replicates within the cytoplasm of a eukaryoticcell. The purified DNA of this virus is not infectious and cannot beused per se to produce vaccinia mutants in a cell in the same manner asSV40. Rather, novel techniques involving the mutation of wild typevaccinia with foreign DNA in vivo within a cell must be employed.

An unpublished paper of the applicants, together with Eileen Nakano(Nakano et al., Proc. Nat'l Acad. Sci. USA 79 1982 1593-1596), reports ademonstration of marker rescue in vaccinia virus. According to theseexperiments, that portion of the L-variant DNA which is normally absentfrom the S-variant can be reintroduced into the S-variant ("rescued")under appropriate conditions. Namely, eukaryotic cells are treated withlive infectious S-variant vaccinia virus together with non-infectiousrestriction fragments of the DNA of the L-variant, representing DNA"foreign" the S-variant, of a particular structure. Namely, that portionof the L-variant DNA which is to be rescued must be present within a DNAchain having portions co-linear with the DNA chain of the S-fragmentinto which it is to be introduced. That is, the "foreign" DNA to beintroduced into the S-variant has, at both ends of the DNA chain, aregion of DNA which is homologous with corresponding sequences in theS-variant. These homologous sequences can be viewed as "arms" attachedto the region of L-variant DNA which is to be rescued by the S-variant.

The mechanism of this recombination is complex and has not yet beenaccomplished in vitro. Apparently, the recombination of the L-DNA intothe S-variant involves homologous base pairing in segments surroundingthe area deleted from the S-variant. Most likely, cross-overs from onestrand of DNA to another result in an in vivo recombination of the DNAto rescue the deleted portion.

This technique of in vivo recombination can be used to introduce foreignDNA other than vaccinia DNA into either the S- or the L-variant ofvaccinia. Thus, the modified Hind III F-fragment incorporating the BamHSV TK fragment therein as DNA "foreign" to vaccinia can be introducedinto vaccinia by treating eukaryotic cells with the modified F-fragmenttogether with infectious L- and/or infectious S-variants of vacciniavirus. In this instance, the portions of the F-fragment flanking the BamHSV TK fragment function as the "arms" mentioned earlier, comprising DNAhomologous with DNA present in the L- or S-variant into which themodified F-fragment is to be introduced. Again, by in vivo processeswithin the cell, the mechanisms of which are not known in detail, theHSV TK-modified F-fragment is incorporated into the vaccinia variants inthe cell and is then capable of replication and expression undervaccinia control.

This in vivo recombination technique is broadly applicable to theintroduction of still other "foreign" DNA into vaccinia, providing apathway by which the genome of vaccinia can be modified to incorporate awide variety of foreign genetic material thereinto, whether such foreignDNA be derived from vaccinia itself, be synthetic, or be derived fromorganisms other than vaccinia.

A wide variety of cells can be used as the host cells in which the invivo recombination described above takes place. The recombination,however, occurs with differing efficiency depending on the cellemployed. Of the cells investigated to date, baby Syrian hamster kidneycells [BHK-21 (Clone 13) (ATCC No. CCL10)] have proved most efficientfor the recombination procedure. However, other cells including CV-1(ATCC No. CCL70), a green monkey kidney cell line, and human (line 143)TK-cells, a 5'-BUdR resistant mutant derived from human cell lineR970-5, have also been infected in this manner to generate vacciniamutants.

These cells are suitably treated with vaccinia and the foreign DNA to beincorporated into the vaccinia while, for convenience, the cells are inthe form of a monolayer. For purposes of in vivo recombination, thecells may be infected with vaccinia followed by treatment with theforeign DNA to be incorporated thereinto, or may first be contacted withthe foreign DNA followed by infection with vaccinia. As a thirdalternative, the vaccinia and foreign DNA may be simultaneously presentat the time the cells are treated.

The viruses are suitably contacted with the cell monolayer while presentin a conventional liquid medium, such as phosphate buffered saline,Hepes buffered saline, Eagle's Special medium (with or without serumaddition), etc., which is compatible with these cells and the viruses.

The foreign DNA is conveniently used to treat these cells while in theform of a calcium phosphate precipitate. Such techniques for introducingDNA into cells have been described in the prior art by Graham et al.,Virology 52, 456-467 (1973). Modifications of the technique have beendiscussed by Stow et al., J. Gen. Virol. 33, 447-458 (1976) and Wigleret al., Proc. Natl. Acad. Sci. USA 76, 1373-1376 (1979). The treatmentstaught in these papers conveniently proceed at room temperature, buttemperature conditions can be varied within limits preserving cellviability, as can the time for which the cells are treated with thevirus and/or foreign DNA precipitate, with various efficiencies of thein vivo recombination. The concentration of the infecting vaccinia virusand the amount of foreign DNA precipitate employed will also affect therate or degree of recombination. Other factors such as atmosphere andthe like are all chosen with a view to preserving cell viability.Otherwise, as long as the three necessary components (cell, virus, andDNA) are present, in vivo recombination will proceed at least to someextent. Optimization of the conditions in a particular case is wellwithin the capabilities of one skilled in the microbiological arts.

Following this recombination step, those vaccinia viruses which havebeen mutated by in vivo recombination must be identified and separatedfrom unmodified vaccinia virus.

Vaccinia viruses mutated by in vivo recombination of foreign DNAthereinto can be separated from unmodified vaccinia virus by at leasttwo methods which are independent of the nature of the foreign DNA orthe ability of the mutant to express any gene which may be present inthe foreign DNA. Thus, first, the foreign DNA in the mutant genome canbe detected by restriction analysis of the genome to detect the presenceof an extra piece of DNA in the mutated organism. In this method,individual viruses isolated from purified plaques are grown and the DNAis extracted therefrom and subjected to restriction analysis usingappropriate restriction enzymes. Again, by detecting the number andmolecular weight of the fragments determined, the structure of thegenome prior to restriction can be deduced. However, because of thenecessity of growing purified plaques, the number of analyses which mustbe made, and the possibility that none of the plaques grown and analyzedwill contain a mutant, this technique is laborious, time consuming anduncertain.

Further, the presence of foreign DNA in vaccinia virus can be determinedusing a modification of the technique taught by Villarreal et al. inScience 196, 183-185 (1977). Infectious virus is transferred from viralplaques present on an infected cell monolayer to a nitrocellulosefilter. Conveniently, a mirror-image replica of the transferred viruspresent on the nitrocellulose filters is made by contacting a secondsuch filter with that side of the first nitrocellulose filter to whichthe viruses have been transferred. A portion of the viruses present onthe first filter is transferred to the second filter. One or the otherof the filters, generally the first filter, is now used forhybridization. The remaining filter is reserved for recovery ofrecombinant virus therefrom once the locus of the recombinant virus hasbeen detected using the hybridization technique practiced on thecompanion, mirror-image filter.

For purposes of hybridization, the viruses present on the nitrocellulosefilter are denatured with sodium hydroxide in a manner known per se. Thedenatured genetic material is now hybridized with a radio-labelledcounterpart of the gene whose presence is sought to be determined. Forexample, to detect the possible presence of vaccinia mutants containingthe Bam HSV TK fragment, the corresponding radio-labelled Bam HSV TKfragment containing ³² P is employed, much in the same manner asdiscussed earlier herein with respect to the detection of plasmidsmodified by the presence of this fragment. Non-hybridized DNA is washedfrom the nitrocellulose filter and the remaining hybridized DNA, whichis radioactive, is located by autoradiography, i.e. by contacting thefilter with X-ray film. Once the mutated viruses are identified, thecorresponding virus plaques present on the second filter, containing amirror image of the viruses transferred to the first filter, are locatedand grown for purposes of replicating the mutated viruses.

The two methods described above involve an analysis of the genotype ofthe organism involved and, as mentioned earlier, can be used whether ornot any gene present within the foreign DNA incorporated into thevaccinia virus is expressed. However, if the foreign DNA is expressed,then phenotypic analysis can be employed for the detection of mutants.For example, if the gene is expressed by the production of a protein towhich an antibody exists, the mutants can be detected by a methodemploying the formation of antigen-antibody complexes. See Bieberfeld etal. J. Immunol. Methods 6, 249-259 (1975). That is, plaques of theviruses including the suspected mutants are treated with the antibody tothe protein which is produced by the mutant vaccinia genotype. Excessantibody is washed from the plaques, which are then treated with proteinA labelled with ¹²⁵ I. Protein A has the ability of binding to the heavychains of antibodies, and hence will specifically label theantigen-antibody complexes remaining on the cell monolayer. After excessradioactive protein A is removed, the monolayers are again picked up byplaque lifts onto nitrocellulose filters and are subjected toautoradiography to detect the presence of the radio-labelled immunecomplexes. In this way, the mutated vaccinia viruses producing theantigenic protein can be identified.

In the specific instance in which the foreign DNA includes the HSV TKgene, once it is known that the mutated vaccinia virus expresses the HSVTK gene therein, a much simpler and elegant means for detecting thepresence of the gene exists. Indeed, the ease of discrimination betweenvaccinia mutants containing the HSV TK gene and unmodified vaccinia freeof this gene provides a powerful tool for discriminating betweenvaccinia virus mutants containing other exogenous genes either presentalone in the vaccinia genome or present therein in combination with theHSV TK gene. These methods are described more in detail later herein.

Since eukaryotic cells have their own TK gene and vaccinia virussimilarly has its own TK gene (utilized, as noted above, for theincorporation of thymidine into DNA), the presence and expression ofthese genes must be in some way distinguished from the presence andexpression of the HSV TK gene in vaccinia mutants of the type underdiscussion. To do this, use is made of the fact that the HSV TK genewill phosphorylate halogenated deoxycytidine, specificallyiododeoxycytidine (IDC), a nucleoside, but neither the TK gene ofvaccinia nor the TK gene of cells will effect such a phosphorylation.When IDC is incorporated into the DNA of a cell it becomes insoluble.Non-incorporated IDC, on the other hand, is readily washed out from cellcultures with an aqueous medium such as physiologic buffer. Use is madeof these facts as follows to detect the expression of the HSV TK gene invaccinia mutants.

Namely, cell monolayers are infected with mutated virus under conditionspromoting plaque formation, i.e. those promoting cell growth and virusreplication. When the cells are infected, they are then treated withcommercially available radio-labelled IDC (IDC*), labelling being easilyeffected with ¹²⁵ I. If the cells are infected with a virus containingthe HSV TK gene, and if the HSV TK gene present therein is expressed,the cell will incorporate IDC* into its DNA. If the cell monolayers arenow washed with a physiologic buffer, non-incorporated IDC* will washout. If the cell monolayers are next transferred to a nitrocellulosefilter and exposed to X-ray film, darkening of the film indicates thepresence of IDC* in the plaques and demonstrates the expression of theHSV TK gene by the vaccinia mutants.

Using the aforementioned genotypic and phenotypic analyses, theapplicants have identified two vaccinia mutants denominated VP-1 andVP-2. VP-1 (ATCC No. VR 2032) is a recombinant vaccinia virus derivedfrom vaccinia S-variant modified by in vivo recombination with theplasmid pDP 132. VP-2 (ATCC No. VR 2030) is an S-variant vaccinia virusmodified by recombination with pDP 137.

FIG. 4A is a Hind III restriction map of the vaccinia genome showing thesite of the HSV TK gene insertion. FIGS. 4B and 4C magnify the Hind IIIF-fragment respectively contained in VP-1 and VP-2 to show theorientation of the Bam HI HSV TK fragment therein. Attention is calledto the fact that the in vivo recombination of pDP 137 with the S-variant(i.e. VP-2 ) effects deletion of one of the Bam HI HSV TK fragmentspresent in tandem in the starting plasmid.

As mentioned earlier, the fact that the HSV TK gene is expressed can beused for a rapid and easy detection and identification of mutants whichcontain or are free of HSV TK gene or of a foreign gene present alone orin combination with the HSV gene. The test and its bases are describedimmediately below.

The applicants have isolated, in biologically pure form, a vacciniamutant, an S-variant in particular, which is free of anynaturally-occurring functional TK gene, denominated VTK⁻ 79 (ATTC No. VR2031). Normally, the S- and L-variants discussed earlier herein have aTK gene in the Hind III fragment J thereof. If this mutant, free ofvaccinia TK gene activity, is used for the production of further mutatedorganisms containing the HSV TK gene, incorporated into the vacciniamutant by the techniques described earlier herein, the HSV TK genepresent in such resultant mutants will be the only functional TK genepresent in the virus. The presence or absence of such an HSV TK gene canbe immediately detected by growing cells infected with the viruses onone of several selective media.

Namely, one such selective medium contains bromodeoxyuridine (BUdR), anucleoside analogous to thymidine, but highly mutagenic and poisonous toorganisms such as a cell or virus when present in DNA contained therein.Such a medium is known from Kit et al., Exp. Cell Res. 31, 297-312(1963). Other selective media are the hypoxanthine/aminopterin/thymidine(HAT) medium of Littlefield, Proc. Natl. Acad. Sci. USA 50, 568-573(1963) and variants thereof such as MTAGG, described by Davis et al., J.Virol. 13, 140-145 (1974) or the further variant of MTAGG described byCampione-Piccardo et al. in J. Virol. 31, 281-287 (1979). All thesemedia selectively discriminate between organisms containing andexpressing a TK gene and those which do not contain or express any TKgene. The selectivity of the media is based on the following phenomena.

There are two metabolic pathways for the phosphorylation of thymidine.The primary metabolic pathway does not rely upon thymidine kinase and,while it synthesizes phosphorylated thymidine by intermediatemechanisms, it will not phosphorylate BUdR or directly phosphorylatethymidine. The secondary metabolic pathway does involve the activity ofthymidine kinase and will result in the phosphorylation of boththymidine and its analog, BUdR. Since BUdR is a poisonous highlymutagenic substance, the presence of TK, such as the HSV TK underdiscussion, in an organism will result in the phosphorylation of BUdRand its incorporation into the DNA of the growing organism, resulting inits death. On the other hand, if the TK gene is absent or not expressed,and the primary metabolic pathway which then is followed results in thesynthesis of phosphorylated thymidine, but not in the phosphorylation ofBUdR, the metabolizing organism will survive in the presence of BUdRsince this substance is not incorporated into its DNA.

The growth behaviors discussed above are summarized in FIG. 5 of theaccompanying drawings tabulating the growth behavior of organismsexpressing TK (TK⁺) and organisms free of or not expressing the TK gene(TK⁻) on a normal medium, on a selective medium such as HAT which blocksthe primary metabolic pathway not using TK, and on a medium containingBUdR. TK⁺ and TK⁻ organisms will both grow on a normal growth medium byemploying the primary metabolic pathway not requiring TK. On a selectivemedium such as HAT which blocks the primary metabolic pathway notrelying on TK, the TK⁺ organism will nevertheless grow because theenzyme accomplishes the phosphorylation necessary for incorporation ofthymidine into DNA. On the other hand, the TK⁻ organisms will notsurvive. In contrast, if the organisms are grown on a medium containingBUdR, the TK⁺ variants will die since TK phosphorylates BUdR and thispoisonous material is incorporated in the DNA. In contrast, since BUdRis not phosphorylated by the primary metabolic pathway, the TK⁻ variantwill grow since BUdR is not incorporated into the DNA.

Thus, if a vaccinia virus free of vaccinia TK, such as VTK⁻ 79, is usedas the vaccinia virus into which the HSV TK gene is inserted by thetechniques of the present invention, the presence and expression, or theabsence, of the HSV TK gene therein can be readily determined by simplygrowing the recombinants on a selective medium such as HAT. Thoseviruses which are mutated will survive since they use the HSV TK tosynthesize DNA.

The applicants have indeed prepared several mutants of vaccinia virusfree of vaccinia TK. These have been denominated VP-3 (ATCC No. VR2036), a recombinant of VTK⁻ 79 and pDP 132, and VP-4 (ATCC No. VR2033), a recombinant of VTK⁻ 79 and pDP 137. The latter expresses theHSV gene and can readily be identified using the selective mediamentioned above.

Two additional recombinant viruses, denominated VP-5 (ATCC No. VR 2028),and VP-6 (ATCC No. VR 2029), are respectively recombinants of pDP 132and pDP 137 with VTK⁻ 11 (ATCC No. VR 2027), a known L-variant ofvaccinia which does not express the vaccinia TK gene. Thus, DNA can beintroduced in excess of the maximum vaccinia genome length.

The techniques of the present invention can be used to introduce the HSVTK gene into various portions of the vaccinia genome for purposes ofidentifying non-essential portions of the genome. That is, if the HSV TKgene can be inserted into the vaccinia genome, as it is in the Hind IIIF-fragment thereof, the region of the genome into which it has beenintroduced is evidently non-essential. Each non-essential site withinthe genome is a likely candidate for the insertion of exogenous genes sothat the methods of the present invention are useful in mapping suchnon-essential sites in the vaccinia genome.

Further, if the HSV TK gene is coupled with another exogenous gene andthe resultant combined DNA material is put into a vaccinia virus free ofvaccinia TK gene, such as VTK⁻ 79, recombinants which are formed andwhich contain the foreign gene will express the HSV TK gene and can bereadily separated from the TK⁻ variants by the screening techniquedescribed immediately above.

A further embodiment of the invention involves the preparation of avaccinia Hind III F-fragment containing an exogenous gene therein andthe treatment of cells with the fragment together with a vaccinia mutantnot expressing the vaccinia TK gene but having the HSV TK geneincorporated therein by in vivo recombination according to thetechniques of the present invention. As with the marker rescue mentionedearlier herein, and the in vivo techniques employed to incorporate theTK-modified Hind III F-fragment into vaccinia, cross-over andrecombination can occur to produce a further mutant in which the HSV TKmodified F-fragment is replaced by an F-fragment containing anotherexogenous gene. The resulting vaccinia mutant, in which the HSV TKF-fragment has been replaced by an F-fragment containing the exogenousgene, will be totally free of TK, whereas the non-mutated parent viruspredominantly present will still be HSV TK⁺. Similarly a foreign genemay be inserted into the HSV TK gene present in such a vaccinia mutant,disrupting the integrity of the gene rendering the recombinant organismTK⁻ in comparison with the non-mutated TK⁺ parent. In both instances, animmediate discrimination can be made between those vaccinia mutantscontaining the foreign gene and those which are free of any TK by growthon BUdR and/or a special medium such as HAT.

FIGS. 7A-C can best be understood in conjunction with FIGS. 3A-C. Thus,it will be seen from FIG. 3B that plasmid pDP 3, prior to theincorporation of any additional DNA therein, has a molecular weight of11.3 megadaltons (md). When further DNA is incorporated therein, such asthe herpes Bam TK fragment shown in FIG. 3B, to produce plasmids pDP 132and pDP 137, the latter plasmids have an increased molecular weight of,respectively, 13.6 and 15.9 md. Since these molecular weights areapproximately at the upper limit of replication for the plasmid, it hasproved desirable to create a plasmid containing the vaccinia Hind IIIF-fragment, but which plasmid is of a lower molecular weight than pDP 3shown in FIG. 3B. A method for creating such a lower molecular weightplasmid is shown in FIGS. 7A-C.

More in particular, FIG. 7A shows plasmid pDP 3 containing the Hind IIIF-fragment of vaccinia of molecular weight 8.6 megadaltons. As shown inthe Figure, the plasmid contains three sites susceptible to cleavage bythe restriction enzyme Pst I. Two of these sites are within theF-fragment portion of the plasmid, while the third is within thatportion of the plasmid which is derived from parent plasmid pBR 322. Asfurther shown in FIG. 7A, when plasmid pDP 3 is cleaved with Pst I,three fragments are obtained. The fragment, which is solely a portion ofthe vaccinia Hind III F-fragment, has a molecular weight of 3.7 md.There are also two other fragments each combining portions of the parentpBR 322 and vaccinia Hind III F-fragment.

The largest, "pure" F-subfragment, can be easily isolated. As shown inFIG. 7B, the fragment can then be introduced into pBR 322 at a Pst Isite therein after cleavage of the pBR 322 plasmid with Pst I. Thejoinder of the parent fragments with T₄ DNA ligase produces the newplasmid pDP 120, shown in FIG. 7C, which has a molecular weight of only6.4 md. The lower molecular weight of the pDP 120 plasmid, in comparisonwith pDP 3, permits the introduction thereinto of longer DNA sequenceswithout approaching the upper limit of replication as do plasmids pDP132 and pDP 137 shown in FIG. 3C.

Again, a better understanding of FIGS. 8A-D will be had by referring toFIGS. 3A-C. More in particular, FIGS. 3B and 3C show the incorporationof a herpes Bam TK fragment into plasmid pDP 3 to form plasmids pDP 132and 137. As explained more in detail in Example X of the application,this herpes Bam TK fragment is introduced into vaccinia virus by an invivo recombination technique involving simultaneous treatment ofsuitable cells with vaccinia virus and Hind III-treated pDP 132 or pDP137. It will be evident from an inspection of FIG. 3C that treatment ofthe aforementioned plasmids with Hind III will excise that portion ofthe plasmids originally derived from plasmid pBR 322, since the herpesBam TK fragment to be incorporated into the vaccinia virus by in vivorecombination was present in a vaccinia Hind III F-fragment joined withthe pBR 322 segment at a Hind III site. Thus, the herpes Bam TK gene isincorporated into vaccinia without the pBR 322 DNA sequence.

However, because of the numerous restrictions sites available in the pBR322 plasmid, for example including Eco RI, Hind III, Bam HI, Pst I,etc., the plasmid is particularly advantageous for the introduction ofDNA sequences thereinto. Hence, it would be desirable to be able tointroduce pBR 322 into a vector such as vaccinia virus.

FIGS. 8A-D show the development of two plasmids by means of which theversatile DNA sequence of pBR 322 can be incorporated into vacciniavirus by in vivo recombination and, particularly, the production of twovaccinia mutants, VP 7 and VP 8, containing the pBR 322 DNA sequence.

More in particular, FIG. 8A shows the vaccinia Hind III F-fragment alsoshown in FIG. 3A of the drawings. The linear segment can be self-ligatedto form a circular F-fragment as also shown in FIG. 8A. The joined HindIII termini are indicated on both the linear and circular fragment as"a" and "d", respectively. The termini on either side of a Bam HI siteare also shown in FIG. 8A as "c" and "b".

As particularly shown in FIG. 8B, this circularized F-fragment can betreated with Bam HI to produce a linear DNA sequence in which the Bam HItermini "b" and "c" are shown with respect to the Hind III termini "a"and "d". This linear sequence will be referred to as an "invertedF-fragment".

If, as further shown in FIG. 8B. Bam HI-treated pBR 322 and the linearinverted F-fragment sequence of FIG. 8B are combined with T₄ DNA ligase,two plasmids are produced, depending on the relative alignment of theinverted F-fragment and the parent pBR 322 sequence. These two plasmidsare shown in FIG. 8C as pDP 301 B and pDP 301 A, each of which has thesame molecular weight of 11.3 md.

The incorporation of plasmids pDP 301 A and 301 B into vaccinia by invivo recombination is shown in FIG. 8D. Namely, each of these plasmidswas incorporated by in vivo recombination into vaccinia virus VTK⁻ 79,respectively to produce vaccinia mutants VP 7 (ATCC No. VR 2042) and VP8 (ATCC No. VR 2053). As shown in this Figure, for this purpose the pDP301 plasmids are each cleaved with Sst I to produce linear DNA sequencesthe termini of which are homologous with a corresponding DNA sequencepresent in the F-portion of the vaccinia virus genone. Simultaneoustreatment of cells with the Sst I-treated plasmids and vaccinia virusresults in in vivo recombination with incorporation of the pBR 322 DNAsequence into the virus genome.

The advantage of the presence of the pBR 322 sequence in the vacciniagenome of VP 7 and VP 8 is that in vivo recombination can be readilyeffected using these variants and pBR 322 sequences modified to have avariety of foreign DNA sequences therein. In this instance, it is thehomologous base pairs of pBR 322 in the vaccinia genome and in themodified pBR 322 DNA sequence to be introduced which facilitatecrossover and recombination, as is illustrated hereinafter with respectto the construction of further new vaccinia virus mutants identified asVP 10, VP 13, VP 14, and VP 16.

FIGS. 9 and 10 concern the insertion of an influenza gene into vacciniato provide two further vaccinia mutants, VP 9 and VP 10.

The influenza genome consists of eight separate pieces of RNA each ofwhich codes for at least one different protein. One of the principalimmunogenic proteins is the hemagglutinin protein and because of thisthe HA gene was chosen for insertion into vaccinia. The genome of theinfluenza virus contains genes in an RNA sequence and, for incorporationinto a plasmid, they must be converted into a DNA copy, identified ascDNA. As known in the art, the cDNA copy of the HA RNA genome is madeusing reverse transcriptase, all as described by Bacz et al. in NucleicAcids Research 8, 5845-5858 (1980).

The influenza virus exists in a number of variants, classified accordingto the nature of the HA gene and another of the eight genes, namely thatcoding for neuraminidase. Within the influenza virus family, there arethree main types of the HA serotype, designated H1-H3.

In the construction of vaccinia virus mutant VP 9 and 10, the influenzavirus employed was A/PR/8/34, which contains an H1 HA gene.

FIG. 9A shows two circular plasmids, pJZ 102 A and pJZ 102 B. Theplasmids were prepared by incorporating a cDNA copy of the influenzahemagglutinin (HA) gene into pBR 322 at the Hind III site. The A and Bplasmid variants differ in the orientation of the HA gene therein, ashas been indicated in FIG. 9A by reference to an initiation codoncontained within the HA gene and located within the gene by itsproximity to an Ava I site within the gene. The relative positions ofthe Ava I site and the initiation codon in the pJZ 102 A and B variantsare indicated in FIG. 9A.

If plasmid pJZ 102 A is treated with Bam HI and T₄ DNA ligase in thepresence of an "inverted" F-fragment of vaccinia virus (the latter shownin FIG. 8B), the result is a further plasmid shown in FIG. 9B as pJZ 102A/F in which the pJZ 102 A parent plasmid is combined with the vacciniaF-fragment.

As further shown in FIG. 9B if the pJZ 102 A/F plasmid shown in FIG. 9Bis incorporated into the VTK⁻ 79 strain of vaccinia virus by in vivorecombination, a vaccinia mutant, VP 9 (ATCC VR No. 2043), is produced,which mutant contains and expresses the influenza hemagglutinin antigen(HA) gene and can be used, as hereinafter described, to promote theproduction of antibodies to the antigen in a mammal.

FIG. 9C is a map of that portion of the genome of VP 9 containing thepJZ 102 A/F DNA sequence and the HA gene therein.

FIGS. 10A-C show the construction of a second vaccinia mutant containingthe influenza hemagglutinin (HA) gene, which vaccinia mutant isdesignated herein as VP 10 (ATCC No. VR 2044).

More in particular, the VP10 mutant is constructed by in vivorecombination of DNA from plasmid pJZ 102 B (cf. FIG. 9A) with the pBR322 DNA sequence found in vaccinia mutant VP 7, the production of whichmutant from plasmid pDP 301A is shown in FIGS. 8C and 8D.

Thus, FIG. 10A is a linear DNA map of pJZ 102B after treatment of thatcircular plasmid with Bam HI. Again, the initiation codon within the HAgene is indicated with reference to an Ava I site within the gene whichis, in turn, contained within the DNA sequence of plasmid pBR 322. FIG.10B shows a portion of the genome found within vaccinia mutant VP 7 asthe result of the incorporation of plasmid pDP 301A into VTK⁻ 79 by invivo recombination. More in particular, FIG. 10B shows the presence ofthe pBR 322 genome surrounded on each side by portions of the F-fragmentof vaccinia virus, the presence of which F "arms" permitted theincorporation of the pBR 322 DNA sequence into the vaccinia genome inthe first place.

Reference has been made earlier in the specification to using in vivorecombination to render an HSV TK⁺ vaccinia virus HSV TK⁻. This involvesreplacing the HSV TK gene, present in such a virus, with an HSV TK genecontaining a foreign gene therein rendering the HSV gene TK⁻. The workunder discussion illustrates such a technique using other DNA,specifically pBR 322 DNA, which also is exogenous to vaccinia. That is,recombination will occur with vaccinia so long as there are homologoussequences, in the transfecting (donor) DNA and in the infecting virus,flanking the foreign gene to be inserted, whether such sequences are orare not endogenous vaccinia sequences. Still other DNA sequences can beinserted into vaccinia and subsequently utilized for in vivorecombination in a similar fashion.

Because of the presence of homologous base pairs in the pBR 322 portionsof pJZ 102B (shown in FIG. 10A) and the pBR 322 DNA sequence containedwithin the genome of VP 7 (cf. FIG. 10B), crossover can occur during invivo recombination involving the simultaneous treatment of cells with VP7 and pJZ 102B, with the incorporation of the HA fragment into thevaccinia genome to create the vaccinia mutant VP 10, as shown in FIG.10C.

VP 10 illustrates that recombination in vivo can occur within the 350base pairs between the Hind III and Bam HI sites at the right end of thepBR 322 sequences shown in FIGS. 10A-C.

To determine whether or not viruses VP 9 and VP 10 are expressing the HAgene, a series of tissue cultures was prepared. Namely, BHK cells,present in a first pair of Petri dishes, were infected with A/PR/8/34influenza virus. CV-1 cells, present in another pair of Petri disheswere infected with VP 9 vaccinia variant, and CV-1 cells present in athird pair of Petri dishes were infected with the VP 10 vacciniavariant. After permitting the viruses to grow within the cells, theinfected cells present in one of each of the three pairs of Petri disheswere treated with H1 HA antiserum: the second set of three cell cultures(one BHK and two CV-1 cultures) were treated with H3 HA antiserum. Allof the cell cultures were next washed and then treated with protein Alabelled with ¹²⁵ I. After treatment with the labelled protein A, thecell cultures are again washed and then radioautographed. If influenzaantigen is being produced by the infected cells, the antigen will reactwith antibodies contained within the H1 HA antiserum but not the. H3 HAantiserum. These complexes will not be washed from the plates and the¹²⁵ I protein A will bind with the constant portion of the heavy chainsof the residual antibody in the complex if the complex is present.

It was determined that complexes were formed in the Petri dish infectedwith A/PR/8/34 and treated with H1 HA antiserum, as was also true of theCV-1 cells infected with VP 9. In contrast, no antigen-antibodycomplexes were formed in any cells infected with VP 10, nor was anycomplex formation detected in the cell cultures infected either withA/PR/8/34 or VP 9 when these cell cultures were treated with H3 HAantiserum.

From this experiment, it can be concluded, first, that the VP 10vaccinia variant does not express the H1 HA gene at a level detectibleby this assay. Conversely, the VP 9 variant does express this gene.Further, the expression VP 9 is specific to H1 HA, since there is nocomplex formation in the VP 9-treated cell cultures which aresubsequently contacted with H3 HA antiserum.

Knowing that VP 9 expresses the H1 HA gene in vitro, it was next testedwhether the VP 9 vaccinia mutant would sufficiently express the H1 HAgene to stimulate the formation of antibodies in an animal infected withthis vaccinia mutant.

For this tests rabbits were infected with vaccinia virus VP 9 byintravenous injection. After 17, 25, and 41 days blood was withdrawnfrom the rabbits and the serum was collected. The presence of antibodiesto H1 HA within this inhibited red blood cell agglutination. Thisindicated the presence, in the antiserum, of H1 HA antibodies in anamount in excess of the HA antigen added thereto.

These experiments demonstrate two important facts. First, it is possibleto create a vaccinia mutant according to the techniques of the presentinvention, which mutant when introduced into an animal model willstimulate the production, even with only primary infection, of antibodyto a protein coded for by a gene within the vaccinia mutant, which geneis foreign both to vaccinia and to the animal into which it isintroduced. Second, the experiments show that the production ofantibodies by the animal to vaccinia itself does not interfere with thesimultaneous production of antibodies to the product coded for by theforeign DNA contained within the vaccinia mutant.

The construction of plasmids pDP 250A and pDP 250B and theirincorporation into VTK⁻ 79 to give, respectively, new vaccinia mutantsVP 12 (ATCC No. VR 2046) and VP 11 (ATCC No. VR 2045) is shown in FIGS.11A-E.

It is possible to isolate a circular DNA comprising the entire hepatitisB virus (HBV) genome. As shown in FIG. 11A, the genome is represented ascomprising a region coding for the surface antigen, including apre-surface antigen region containing an Eco RI site therein. Thesesurface antigen regions are depicted in FIG. 11A as a "block" containedwithin the genome, the remaining DNA of serum was tested by a series ofin vitro experiments similar to those earlier described involving theinfection of cell cultures with A/PR/8/34 and VP 9. The formation of animmune complex was observed when a cell layer infected with VP 9 wastreated with the rabbit antiserum. However, this test merely indicatesthat the rabbit produced antibodies to the vaccinia virus: it is notpossible to determine whether antibodies were produced specifically tothe H1 HA antigen. However, the formation of a complex between therabbit antiserum and a BHK cell monolayer infected with A/PR/8/34 didindicate the presence, in the antiserum, of antibodies specific to theH1 HA antigen.

As a separate criterion for the production of HA antibodies, anhemagglutinin inhibition assay was performed. This test makes use of theproperty of HA to agglutinate red blood cells into large complexes.

To perform the assay, the rabbit antiserum was first serially diluted.Each serial dilution of the antiserum was reacted with the same fixedquantity of hemagglutinin, obtained by extracting cells infected withinfluenza virus. If antibodies are present in the antiserum in an amountequal to or in excess of the amount of hemagglutinin introduced intoeach serial dilution, the resulting mixture will inhibit theagglutination of red blood cells admixed therewith because of thepresence of an excess of antibody with respect to the agglutinatingagent, HA.

In the serial dilution performed (on the 45 day antiserum), alldilutions up to and including 1:320 which is represented by a zig-zagline. When the genome is treated with Eco RI to cleave it, the linearDNA sequence obtained is disrupted in the pre-surface antigen portionthereof such that a portion of the pre-surface antigen region is presentat each terminus of the linear DNA sequence. The two termini of thesequence, one on each side of the Eco RI site in the circular genome,are represented both in the circular genome and the linear DNA fragmentrespectively by a circle and square.

If the HBV DNA fragment is now incorporated into pBR 322, as shown inFIG. 11B, and that plasmid which contains two hepatitis B fragments intandem is isolated, the known plasmid pTHBV 1, shown in FIG. 11B, isobtained. This plasmid will contain therein the reconstructedpre-surface antigen and surface antigen regions of the originalhepatitis B genome, as has been pointed out by encircling these regionswith dashed lines in the depiction given of plasmid pTHBV 1 in FIG. 11B.This entire construction is described by Hirschman et al, Proc. Natl.Acad. Sci. USA 77, 5507-5511 (1980).

This sAg region of pTHBV 1 can be isolated by treatment of the plasmidwith the restriction enzyme Bgl II. The pBR 322 portion of the pTHBV 1plasmid contains no Bgl II site, while each of the two HBV DNAfragments, present in tandem in the plasmid contains three Bgl II sites.Thus, pTHBV 1 contains six Bgl II sites, all in the HBV DNA, but all ofwhich are outside the sAg region. Thus, as shown in FIGS. 11B and 11C,cleavage of pTHBV 1 with Bgl II will produce a linear DNA fragmentcontaining the sAg region of the hepatitis B virus. [Galibert et al.,Nature 281, 646-650 (1979)].

This fragment can be incorporated into the plasmid pDP 120, theproduction of which is earlier described in FIGS. 7A-C, although the pDP120 plasmid contains no Bgl II site.

The recognition sequence for the enzyme Bgl II is -AGATCT- with thecleavage site being between -A and GATCT-. On the other hand, therecognition site for Bam HI is -GGATCC-, with the cleavage site beingbetween -G and GATCC-. Thus, if DNA containing either of theserecognition sites is respectively cut with Bgl II or Bam HI, in eachcase a -GATC- "sticky end" will be produced, which ends will beligatable (but then no longer subject to cleavage by either Bgl II orBam HI).

The new plasmids pDP 250 A and pDP 250 B are constructed as shown inFIGS. 11C and D by partial cleavage of pDP 120 with Bam HI and ligationof the "sticky ends" so produced with the corresponding "sticky ends" ofthe Bgl II fragment of the hepatitis virus genome.

As known in the art, it is possible to discourage the re-circularizationof Bam HI-cleaved pDP 120 by treatment of the cleaved plasmid withalkaline phosphatase, an enzyme which removes terminal phosphoric acidgroups from the 5'-cleaved ends of the linear DNA of pDP 120. Removal ofthe phosphoric acid groups prevents a re-circularization reaction of thepDP 120, but does not interfere with reaction of the 3'-OH termini ofthe linear pDP 120 DNA with the 5'-phosphate ends present on the Bgl IIfragments, with subsequent circularization to produce plasmids such aspDP 250 A and pDP 250 B. Thus, the statistical probability for thecreation of the latter, tetracycline resistant, plasmids can beincreased with the alkaline phosphatase treatment as described.

Ultimately, plasmids pDP 250 A and pDP 250 B are identified byrestriction analysis using Xho I to determine orientation.

As shown in FIGS. 11D and E, the virus mutants VP 11 and VP 12 arerespectively derived from plasmids pDP 250 B and pDP 250 A by in vivorecombination of these plasmids with VTK⁻ 79 vaccinia virus. Crossoverand recombination occur in the long and short "arms" of the vacciniaF-fragment present in the plasmids. That portion of the plasmids derivedfrom pBR 322 is not incorporated into the virus.

FIG. 12A shows the structure of the pTHBV 1 plasmid, known in the artand shown earlier herein in FIG. 11B. As shown in the Figure, if theplasmid is treated with the restriction enzyme Hha I, two identicalfragments will be obtained containing only that region of the HBV genomecontained within the plasmid which codes for the surface antigen, freeof any pre-surface antigen region. (In fact, there are many Hha Irestriction sites within pTHBV 1, and numerous fragments will beproduced upon digestion with this restriction enzyme. However, thefragment of interest discussed above is the largest of the numerousfragments obtained, and can be readily isolated because of this fact.) Alinear DNA map of this Hha I fragment is also shown in FIG. 12A, withthe further indication of a Bam HI site for purposes of orientation.

If the Hha I fragment of FIG. 12A is treated first with T₄ DNApolymerase and then Hind III linkers are added with T₄ DNA ligase, thefragment can be provided with Hind III sticky ends. As known in the art,T₄ DNA polymerase has both a polymerase activity in the 5'- to3'-direction, as well as exonuclease activity in the 3'- to5'-direction. The two opposing activities will result in the "chewingoff" of 3'-OH ends in the Hha I fragment shown in FIG. 12A until anequilibrium state is reached, with the resultant production of a bluntended DNA fragment. The blunt ended fragment can be treated with HindIII linkers, known in the art, which are essentially decanucleotidescontaining therein the recognition sequence for Hind III.

As shown in the map in FIG. 12B, the resulting fragment will have HindIII sticky ends and can be introduced, as shown in FIG. 12B, into pBR322 by treatment of the latter plasmid with Hind III and T₄ DNA ligase.The resultant plasmid, identified in FIG. 12C, is designated as pDP 252.

Finally, as shown in FIG. 12D, this plasmid can be introduced intovaccinia mutant VP 8 by in vivo recombination to produce new vacciniavariant VP 13 (ATCC No. 2047). Expression of the HBV gene by VP 13 hasnot yet been detected.

FIGS. 13A-C show the production of two further virus mutants, VP 16(ATCC No. VR 2050) and VP 14 (ATCC No. VR 2048), each containing the DNAsequence of herpes virus type I which codes for production of the herpesglycoproteins gA+gB.

More in particular, FIG. 13A is a map of the Eco RI fragment F of herpesvirus type I, strain KOS [Little et al, Virology 112, 686-702 (1981)].As is evident from the Figure, the DNA sequence contains numerous Bam HIsites (indicated as B) within the sequence, including one DNA region 5.1md in length between adjacent Bam HI sites and representing the largestBam HI fragment within the Eco RI fragment under discussion.

As further shown in FIG. 13A, this Eco RI fragment can be introducedinto pBR 322 by treatment with Eco RI and T₄ DNA ligase to produce twonew plasmids, respectively identified as pBL 520 A and 520 B,distinguished by the orientation of the Eco RI fragment therein (asindicated by the Hpa I sites, used for orientation.)

Finally, as shown in FIG. 13C, these plasmids can be introduced intovaccinia virus mutant VP 7 (containing the pBR 322 genome) by in vivorecombination analogous to that discussed for FIGS. 10 and 12, thusproducing vaccinia mutants VP 16 and VP 14 respectively.

Thus, this is a further example of the use of the pBR 322 DNA sequence(rather than the DNA sequence of the vaccinia F-fragment) to effect invivo recombination for the production of further vaccinia virus mutants.Also, vaccinia variants VP 16 and 14 produced by this method are ofinterest in containing more than 20,000 base pairs of foreign DNAincorporated into the genome of vaccinia virus. This represents aminimum upper limit of foreign DNA insertion into vaccinia.

FIGS. 14A-C show the production of two further virus mutants, VP 17 andVP 18, into which have been introduced a Bam HI segment of the herpesvirus type I (strain KOS) Eco RI fragment F. The Eco RI fragment F isshown in FIG. 13A as including this 5.1 megadalton Bam HI fragment G.

FIG. 14A shows this Bam HI fragment, including the location therein oftwo Sst I sites which are asymmetric and are used for orientation.

This Bam HI segment of the herpes virus, which still codes for theproduction of herpes glycoproteins gA+gB [cf. De Luca et al., ibid.], isintroduced into pDP 120 (cf. FIG. 7C) by partial digestion with Bam HIand T₄ DNA ligase, as further shown in FIG. 14A.

As shown in FIG. 14B, two new plasmids, pBL 522 A and pBL 522 B, areobtained, each having a molecular weight of 11.6 megadaltons and eachcontaining the Bam HI fragment G of the herpes virus genome in one oftwo different orientations.

Finally, as shown in FIG. 14C, these plasmids can be introduced intoVTK⁻ 79 by in vivo recombination to produce vaccinia mutants VP 17 (ATCCNO. 2051) and VP 18 (ATCC No. VR 2052). Expression of the herpesglycoprotein gene by mutants VP 17 and 18 has not yet been determined.

FIGS. 15A-F illustrate the construction of a further vaccinia variant,VP 22 (ATCC No. VR 2054) wherein foreign DNA is present in the vacciniagenome in a non-essential region different from the F-fragment utilizedfor the production of other vaccinia variants described herein.

More in particular, FIG. 15A shows the Ava I H-fragment of the vacciniavirus L-variant genome. As shown in FIG. 6A, the Ava I fragment isentirely within the region deleted from the S-variant and, hence, isknown to be non-essential for the viability of the virus.

As shown in FIG. 15A, this Ava I H-fragment is combined with a HindIII-cleaved pBR 322 plasmid to form a new plasmid, pDP 202, shown inFIG. 15B. The ligation is accomplished by "blunt-ending" both the Ava IH-fragment of vaccinia and the termini of the Hind III-cleaved pBRplasmid, using T₄ DNA polymerase. When the blunt-ended DNA sequences arecombined in the presence of T₄ DNA ligase, pDP 202 is formed.

As further suggested in FIG. 15B, the new plasmid, pDP 202, is combinedwith a herpes Bgl/Bam TK fragment. The latter fragment is obtained fromthe herpes Bam TK DNA fragment (cf. FIG. 3B) by treatment with Bgl II.The treatment with Bgl II removes the endogenous herpes promoter regioncontained within the Bam TK fragment.

Because, as earlier noted, Bgl II and Bam HI produce the same "stickyends" on DNA treated therewith, the resulting herpes Bgl/Bam TK fragmentcan be inserted into a Bam site within pDP 202.

As suggested by FIG. 15C, such an insertion is effected by partialdigestion with Bam HI and treatment with T₄ DNA ligase.

Since the H-fragment of vaccinia present in pDP 202 contains three BamHI sites, a total of six plasmids can be produced by insertions in thisregion, namely two variants for each of the three Bam HI sites,depending on the orientation in each site of the herpes Bgl/Bam TK DNAsequence. The orientation of the latter can be recognized by thepresence therein of a non-symmetric Sst I site near the Bgl II end ofthe fragment.

As shown in FIG. 15C, two plasmids, pDP 202 TK/A and pDP 202 TK/D areobtained when the herpes Bgl/Bam TK fragment is inserted in the first ofthe three Bam HI sites present within the H-fragment of vaccinia presentin pDP 202. Similarly, two other plasmids, pDP 202 TK/E and /C areobtained upon insertion of the Bgl/Bam DNA sequence in the second of thethree available sites. Finally, two further plasmids, pDP 202 TK/B and/F are obtained upon insertion of the Bgl/Bam fragment in each of twopossible orientations in the third Bam HI site. Of these plasmids, pDP202 TK/E has proved of particular interest.

The plasmid is shown in greater detail in FIG. 15D, wherein theorientation of the Bgl/Bam fragment is indicated.

The plasmid can be incorporated by in vivo recombination into the genomeof VTK⁻ 79 L. FIG. 15E is an Ava I map of the left-hand portion of thisvaccinia genome. A map of the modified genome, which is the genome of VP22, is shown in FIG. 15F.

This vaccinia variant is of particular interest since it shows a higherlevel of TK expression than do variants VP 2, VP 4, and VP 6, in whichthe Bam TK fragment is present within the F-fragment of vaccinia.Further, VP 22 demonstrates the introduction of foreign DNA intonon-essential portions of the vaccinia genome other than the F-fragmentwhich has been used, as a matter of convenience, for the constructionsof other vaccinia variants reported herein.

Finally, since all the herpes virus regulatory sequences are deletedfrom the Bgl/Bam herpes virus DNA sequence by treatment with Bgl II, asdescribed earlier herein, the VP 22 vaccinia variant demonstratesconclusively that transcription in this recombinant virus is initiatedby regulatory signals within the vaccinia genome.

FIG. 16 shows the construction of a plasmid, pRW 120, useful in theconstruction of three further vaccinia variants, vP 53, vP 59, and vP60, discussed further below. Both the pDP 3 and pDP 120 plasmids earlierdescribed herein contain two Bam HI sites within the plasmid, one in thevaccinia fragment present in these plasmids and one in that portion ofthe plasmid derived from pBR 322. This necessitates partial Bam HIdigestions when it is sought to clone into the Bam HI site within thevaccinia fragment. pRW 120 is created to eliminate this problem since itis a plasmid with only a single Bam HI site and that is within a Pst Isubfragment of the vaccinia F-fragment present in the new plasmid.

The new plasmid is derived from the known plasmid pBR 325, which iscommercially available and has a Pst I site, an Eco RI site, and a BamHI site, among others. As is evident from FIG. 16, each of these threecleavage sites is associated with resistance to an antibiotic,specifically to ampicillin (Pst I), to chloramphenicol (Eco RI), and totetracycline (Bam HI).

According to the present invention the parent plasmid is modified toremove the Bam HI site by cleavage of pBR 325 with Bam HI, blunt endingthe result linear DNA with T₄ DNA polymerase anddeoxynucleosidetriphosphates (dNTPs), and rejoining the blunt ends inthe presence of T₄ DNA ligase to form the plasmid pBR 325 (Bam X), whichretains only resistance to ampicillin and chloramphenicol.

A Pst I subfragment of the vaccinia Hind III F-fragment, the derivationof which is shown in FIG. 7A, is now introduced into the Pst I site ofpBR 325 (Bam X) to produce the new plasmid, pRW 120. The introduction ofthis subfragment into the Pst I site of pBR 325 (Bam X) destroys theampicillin resistance of the parent plasmid so that pRW 120 is nowsensitive to both ampicillin and tetracycline, but resistant tochloramphenicol. These properties permit its identification andisolation. The sole Bam HI site present within the vaccinia Pst Isubfragment in pRW 120 is the locus for further introduction of DNA,exogenous to vaccinia, for the creation of additional vaccinia virusmutants.

More in particular, as shown in FIG. 17, a Hind III DNA sequencecontaining the influenza HA gene is obtained from plasmid pJZ 102 (cf.FIG. 9A and Example XV). The DNA is blunt ended with T₄ DNA polymerasein the presence of dNTPs and Bam HI linkers are then added, all bytechniques known to those skilled in the art. The Bam HI terminated HAgene is next incorporated into pRW 120 by linearizing the plasmid withBam HI, treating with calf intestine alkaline phosphatase (CIAP), andligating in the presence of T₄ DNA ligase to form the new plasmid, pDP122 B (ATCC 39736).

Using techniques disclosed elsewhere herein, this plasmid was thenincorporated into vaccinia virus VTK⁻ 79 by in vivo recombination toproduce new vaccinia variant vP 53 (ATCC VR 2060). (This plasmid, pDP122 B, is inserted into the vaccinia fragment such that the 5' to 3'direction of transcription of the HA gene is consistent with thedirection of transcription initiated by the vaccinia promotor sequence.)

Expression of the HA gene was detected, as for the aforementionedmutants vP 9 and vP 10, by radioimmunoassays (RIA) and the production ofHA antibodies by the immunization of rabbits. The immune serum soobtained was tested for activity using an hemagglutination inhibitionassay and by plaque reduction assays determining the ability of theserum to neutralize viral infectivity.

The new virus, vP 53, expresses the HA gene more strongly than either vP9 or vP 10.

FIG. 18 shows the construction of a vaccinia virus mutant, vp 59,containing the hepatitis B virus surface antigen (HBsAg). More inparticular, plasmid pDP 252 (ATCC 39735) (cf. FIG. 12C) is used toamplify the hepatitis gene coding for the s-antigen in E. coli usingknown techniques. DNA containing the gene is then isolated from theplasmid with Hind III, the fragment is blunt ended, and Bgl II linkersare affixed. The resulting fragment is now incorporated into the Bam HIsite of pRW 120 (both Bam HI and Bgl II produce GATC-sticky ends) toform the new plasmid pDP 232 B. Using this plasmid, the HBsAg DNAsequence is incorporated into VTK⁻ 79 by in vivo recombination to formvP 59 (ATCC No. VR 2061).

Cells infected with the vP 59 virus express significant levels of HBsAgin vitro. Radioimmunoassays of the infected cells and of the growthmedium showed the excretion by the cells of HBsAg in amounts of 150-200ng in a 24 hour period per 10⁶ cells at a multiplicity of infection ofabout 2 pfu per cell. Since more than 90 percent of the viralinfectivity was cell-associated, the presence of HBsAg in the growthmedium is not due to lysis of the infected cells. However, significantlevels of HBsAg were not detectible by RIA in unfixed cells infectedwith vP 59, suggesting that sAg does not accumulate in the cellmembrane.

Antisera were again prepared by the inoculation of rabbits, as discussedearlier herein for vaccinia mutant vP 9, and antibody production wasconfirmed by RIA. The level of expression of HBsAg by vacciniarecombinant vP 59 is several thousand times greater than that exhibitedby vP 11 (cf. Example XXXII infra. The greater expression is apparentlyassociated with the absence, from the Hha I fragment present in vP 59,of the pre-sAg region of the HBsAg gene. This same Hha I fragment, freeof the pre-sAg region, is used to construct vP 13 (cf. FIG. 12).However, vP 59 differs from vP 13 in that the latter, in addition tocontaining the sAg sequence, additionally contains the DNA of pBR 322.Indeed, the sAg is inserted into a Hind III site within the pBR 322sequence of vP 8 to form vP 13.

FIG. 19 shows the construction of a vaccinia variant, vP 60 (ATCC VR2062), expressing the herpes simplex virus type 1 glycoprotein D(HSVgD).

gD glycoprotein from Herpes virus type 1 and Herpes virus type 2 havetype-common antigenic determinants so that antibodies to these antigenicdeterminants are cross-reactive with the antigens. That is, antibody tothe type 1 virus gD will react with type 2 gD and vice versa. However,since the glycoproteins are not identical in both viruses, antibody toone virus type will not be as effective in neutralizing antigen of theother type.

More in particular, the Eco RI fragment H of HSV type I (strain KOS) wasinserted into the Eco RI site of pBR 322 to produce two new plasmidscollectively identified as pBL 540. The plasmids differ in theorientation of the Eco RI fragment in pBR 322, which orientation,however, is immaterial to the use to which the plasmids are put. Oneplasmid, designated pBL 540A (ATCC, 68574) was cloned in E. coli foramplification of the Eco RI H-fragment. A 2.9 megadalton Sst I fragmentcontaining the HSVgD gene was isolated from pBL 540, as shown in theFigure. After affixation of Bam HI linkers thereto, the DNA sequence wasintroduced into the Bam HI site of pRW 120 (cf. FIG. 16) to produce thenew plasmid pBL 310. Again, after amplification in E. coli, a Hind IIIto Bam HI subfragment, 1.65 md in length and still containing the HSVgDgene, was isolated. This fragment contains the coding sequence for theherpes gD gene free of the endogenous herpes promotor sequence. Watsonet al. Science 218, 381 (1982).

Bam HI linkers were affixed to this subfragment and the subfragment wasintroduced into the Bam HI site in the vaccinia portion of pRW 120 togive the new plasmid pBL 330A. (A number of restriction sites are shownin pBL 330A in both the pBR 325 sequence and the vaccinia F-subfragmentfor orientation. In pBL 310 and pBL 330A, the 5'-to-3' directiontranscription is from right to left relative to the vaccinia genome.)

The coding sequences for HSVgD have been localized to a Sac I (=SstI)DNA fragment contained within the Eco RI DNA fragment H [cf. Lee et al.(1982) J. Virol. 43, 41-49]. Sac I and Sst I are isoschizomersrecognizing the same 6 base pair DNA sequence, but are derived fromdifferent sources.

Using the technique described in Example XVII, preliminary evidence forthe expression of the HSVgD was detected by RIA in unfixed monolayers ofcells infected with vP 60 reacted with antiserum to herpes virus and ¹²⁵I-labelled protein A.

Rabbits, inoculated both intradermally and intravenously with thevaccinia recombinant vP 60, proved that the recombinant was immunogenic.Thus, in a standard plaque-reduction assay similar to that described inExample XVIII, treatment of HSV type 1 with rabbit antiserum obtained3-5 weeks after inoculation considerably decreased HSV infectivity asmeasured by plaque reduction.

Indeed, rabbits simultaneously inoculated with vP-60 and vP-59 (theHBsAg vaccinia recombinant discussed earlier herein) also produced anantiserum reacting with both HBsAg and HSVgD.

Certain mouse strains, known in the art, are highly susceptible to HSVand develop encephalitis in 5-7 days with subsequent high mortality. Theinoculation of such susceptible mice with vP 60 by interperitonealinjection gave protection against subsequent challenge with infectiousHSV. More in particular, three sets of susceptible mice wererespectively inoculated with saline solution, with wild type vacciniavirus, or with the vaccinia virus vP 60 recombinant. After three weeks,the mice were challenged with interperitoneal injections of infectiousHSV type 1 virus. A 100 percent survival rate of the mice inoculatedwith vP 60 shows the development of protective immunity to HSV.

Protective immunity, further, was not only demonstrated by homologouschallenge with HSV type 1, but by heterologous challenge with HSV type2. In a comparison with wild type vaccinia (VTK⁻ 79) as the immnunogen,two groups of mice were inoculated interperitoneally with the vP 60virus or with the wild type virus respectively. Challenge followed sixweeks later with HSV type 2. As compared with mice immunized with thenon-recombinant virus, the mice immunized with vP 60 showed a muchhigher survival rate.

FIG. 20 is a plot of the antibody response in rabbits inoculated withrecombinant vaccinia virus vP 59 expressing the HBsAg gene. Nys:(FG)rabbits were inoculated intradermally at two or three sites with 1.8(10⁷), 1.8 (10⁸), and 3.6 (10⁸) pfu of the recombinant virus asindicated in the Figure. One rabbit was inoculated with 3.6 (10⁸) pfuintravenously. Further rabbits were injected intravenously orintradermally using 1.8 (10⁸) pfu of both recombinants vP 59 and vP 60.The antiserum was collected at weekly intervals and screened forantibodies reactive with HBsAg using a commercially available RIA kit.The antibody levels detected are noted in RIA units per ml of serum onthe ordinate of the graph shown in FIG. 20. (The "AUSAB"radioimmunoassay kit marketed by Abbott Laboratories was used to measureantibody titers to HBsAg. The kit comprises polystyrene beads coatedwith human HBsAg. The specimen to be tested is combined with the beadsand incubated. Antibodies, if present in the sample, are fixed to thesolid phase antigen. When antigen tagged with ¹²⁵ I is added to thebeads, it binds to antibody on the bead creating a radioactiveantigen-antibody-antigen "sandwich". Measurement of the amount ofradioactivity is indicative of the amount of antibody present in thetest sample.)

A better understanding of the present invention and of its manyadvantages will be had by referring to the following specific Examples,given by way of illustration. The percentages given are percent byweight unless otherwise indicated.

EXAMPLE I Isolation of Vaccinia Hind III Fragments from Agarose Gels.

Restriction endonuclease Hind III was purchased from Boehringer MannheimCorp. Preparative digestions of DNA were performed in 0.6 ml of HInd IIIbuffer containing 10 millimolar (mM) Tris-HCl (pH 7.6), 50 mM NaCl, 10mM MgCl₂, 14 mM dithiothreitol (DTT), and 10 micrograms (μg)/ml ofbovine serum albumin (BSA) in which are present 10-20 μg of vaccinia DNAand 20-40 units of Hind III (1 unit is the amount of enzyme sufficientto cleave 1 μg of lambda-DNA completely in 30 minutes.)

Vaccinia DNA was extracted and purified from virions as follows.Purified virions were lysed at a concentration having an optical densityper ml of 50 measured at 260 nanometers (A₂₆₀) in 10 mM Tris-HCl (pH7.8), 50 mM beta-mercaptoethanol, 100 mM NaCl, 10 mM Na₃ EDTA, 1%Sarkosyl NL-97, and 26% sucrose. Proteinase K was added to 100 μg/ml andthe lysate incubated at 37° C. overnight. DNA was extracted by theaddition of an equal volume of phenol-chloroform (1:1). The organicphase was removed and the aqueous phase reextracted until the interfacewas clear. Two additional extractions with chloroform were performed andthe aqueous phase was then dialyzed extensively against 10 mM Tris-HCl(pH 7.4) containing 0.1 mM Na₃ EDTA at 4° C. DNA was concentrated toapproximately 100 μg/ml with Ficoll (a synthetic high copolymer ofsucrose and epichlorohydrin).

Digestion of the DNA was for 4 hours at 37° C. The reactions wereterminated by heating to 65° C. for 10 minutes followed by addition ofan aqueous stop solution containing 2.5% of agarose, 40% of glycerol, 5%of sodium dodecyl sulfate (SDS), and 0.25% of bromophenol blue (BPB).Samples were layered at 65° C. onto agarose gel and allowed to hardenprior to electrophoresis.

Electrophoresis was carried out in 0.8% agarose gels (0.3×14.5×30 cm) inelectrophoresis buffer containing 36 mM Tris-HCl (pH 7.8), 30 mM NaH₂PO₄, and 1 mM EDTA. Electrophoresis was at 4° C. for 42 hours at 50volts. The gels were stained with ethidium bromide (1 μg/ml inelectrophoresis buffer). The restriction fragments were visualized withultraviolet (UV) light and individual fragments were cut from the gel.

Fragments were separated from the agarose gel according to the procedureof Vogelstein et al., Proc. Natl. Acad. Sci. USA 76, 615-619 (1979)using glass powder as follows. The agarose gel containing a DNA fragmentwas dissolved in 2.0 ml of a saturated aqueous solution of NaI. 10 mg ofglass powder were added per μg of DNA calculated to be present. Thesolution was rotated at 25° C. overnight to bind the DNA to the glasspowder. The DNA-glass powder was collected by centrifugation at 2000 rpmfor 5 minutes. The DNA-glass was then washed with 5 ml of 70% NaI. TheDNA-glass was again collected by centrifugation and washed in a mixtureof 50% buffer [20 mM Tris-HC1 (pH 7.2), 200 mM NaCl, 2 mM EDTA] and 50%ethanol. The DNA-glass was collected again by centrifugation and wasgently suspended in 0.5 ml of 20 mM Tris-HCl (pH 7.2), 200 mM NaCl, and2 mM EDTA. The DNA was then eluted from the glass powder at 37° C. byincubation for 30 minutes. The glass was then removed by centrifugationat 10000 rpm for 15 minutes. DNA was recovered from the supernatant byethanol precipitation and dissolved in 10 mM Tris-HCl (pH 7.2)containing 1 mM EDTA.

The F-fragment isolated in this way was used in the following Examples.

EXAMPLE II Insertion of the Vaccinia Hind III-F Fragment Into the HindIII Site of pBR 322 (Construction of pDP 3 [pBR 322- Vaccinia Hind III

Vaccinia Hind III-F fragment was isolated from preparative agarose gelsas described in Example I. This fragment was inserted into the Hind IIIsite of pBR 322 [Bolivar et al., Gene, 2, 95-113 (1977)] as follows.

Approximately 200 nanograms (ng) of pBR 322 were cleaved with Hind IIIin 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 10 mM MgCl₂, and 14 EM DTT [HindIII buffer] using 1 unit of enzyme for 1 hour at 37° C. The reaction wasstopped by heating to 65° C. for 10 minutes. 500 ng of isolated Hind IIIvaccinia F-fragment were added and the DNAs co-precipitated with 2volumes of ethanol at -70° C. for 30 minutes. The DNA was then washedwith 70% aqueous ethanol, dried, and resuspended in ligation bufferconsisting of 50 mM Tris-HC1 (pH 7.6), 10 FM MgCl₂, 10 mM DTT, and 1 mMadenosine triphosphate (ATP). Approximately 100 units of T₄ DNA ligase(New England Biolabs) were then added and the mixture was incubated at10° C. overnight. The ligase-treated DNA was then used to transform E.coli HB101 [Boyer et al., J. Mol. Biol. 41, 459-472 (1969)].

EXAMPLE III Transformation of E. coli and Selection for RecombinantPlasmids

Competent cells were prepared and transformed with plasmids according tothe procedure described by Dagert et al., Gene 6, 23-28 (1979). E. coliHB101 cells were made competent by inoculating 50 ml of LB broth (1% ofbacto-tryptone, 0.5% of bacto-yeast extract, and 0.5% of NaClsupplemented with 0.2% of glucose) with 0.3 ml of an overnight cultureof the cells and allowing them to grow at 37° C. until the culture hadan optical density (absorbence), at 650 nanometers (A₆₅₀ ), of 0.2, asmeasured with a spectrophotometer. The cells were then chilled on icefor 10 minutes, pelleted by centrifugation, resuspended in 20 ml of cold0.1 molar (M) CaCl₂, and incubated on ice for 20 minutes. The cells werethen pelleted and resuspended in 0.5 ml of cold 0.1M CaCl₂ and allowedto remain at 4° C. for 24 hours. The cells were transformed by addingligated DNA (0.2-0.5 mg in 0.01-0.02 ml of ligation buffer) to competentcells (0.1 ml). The cells were then incubated on ice for 10 minutes andat 37° C. for 5 minutes. 2.0 ml of LB broth were then added to the cellsand incubated at 37° C. for 1 hour with shaking. Aliquots of 10microliters (μl) or 100 μl were then spread on LB agar plates containingampicillin (Amp) at a concentration of 100 μg/ml.

The transformed bacteria were then screened for recombinant plasmids bytransferring ampicillin resistant (Amp^(R)) colonies to LB agarcontaining tetracycline (Tet) at 15 ug/ml. Those colonies which wereboth Amp^(R) and tetracycline sensitive (Tet^(S)) (approximately 1%)were screened for intact vaccinia Hind III-F fragment inserted into pBR322 according to the procedure of Holmes et al., Anal. Bioch. 114,193-197 (1981). 2.0 ml cultures of transformed E. coli were grownovernight at 37° C. The bacteria were pelleted by centrifugation andresuspended in 105 ul of a solution of 8% sucrose, 5% Triton X-100, 50mM EDTA, and 50 mM Tris-HCl (pH 8.0), followed by the addition of 7.5 μlof a freshly prepared solution of lysozyme (Worthington Biochemicals)[10 mg/ml in 50 mM Tris-HCl(pH 8.0)]. The lysates were placed in aboiling water bath for 1 minute and then centrifuged at 10000 rpm for 15minutes. The supernatant was removed and plasmid DNA precipitated withan equal volume of isopropanol. The plasmids were then resuspended in 40μl of Hind III buffer and digested with 1 unit of Hind III for 2 hours.The resulting digests were then analyzed on a 1.0% analytical agarosegel for the appropriate Hind III-F fragment. One such recombinantplasmid containing an intact Hind III-F fragment, denominated pDP 3, wasused for further modification. (See FIG. 3B).

EXAMPLE IV Preparative Isolation of pDP 3

Large scale isolation and purification of plasmid DNA was performed by amodification of the procedure of Clewel et al., Proc. Natl. Acad. Sci.USA 62, 1159-1166 (1969). 500 ml of LB broth were inoculated with 1.0 mlof an overnight culture of E. coli HB 101 containing pDP 3. At anoptical density (A₆₀₀) of approximately 0.6, chloramphenicol was added(100 μg/ml) to amplify the production of plasmids [Clewel, J. Bacteriol.110, 667-676 (1972)]. The bacteria were incubated at 37° C. for anadditional 12-16 hours at which time they were collected bycentrifugation at 5000 rpm for 5 minutes, washed once in 100 ml of TENbuffer [0.1 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA], collected bycentrifugation and resuspended in 14 ml of a 25% solution of sucrose in0.05M Tris-HCl (pH 8.0). 4.0 ml of lysozyme solution [5 mg/ml in 0.25MTris-HCl (pH 8.0)] were added and the mixture was incubated at roomtemperature for 30 minutes followed by the addition of 4.0 ml of 0.25MEDTA (pH 8.0). The mixture was then put on ice for 10 minutes. 2.0 ml ofpancreatic RNase A (Sigma Chemical Co.,) [1 mg/ml in 0.25M Tris-HCl (pH8.0)] were added to this mixture, which is then incubated at roomtemperature for 1 minute. The cells were then lysed by adding 26 ml of alytic Triton solution [1% Triton X-100, 0.05M EDTA, 0.05M Tris-HCl (pH8.0)]. The mixture was incubated at room temperature for 30-60 minutes.The lysate was cleared by centrifugation at 17000 rpm for 30 minutes at4° C. The supernatant was then removed and plasmid DNA separated fromchromosomal DNA on dye-bouyant CsCl gradients.

For this purpose, CsCl-ethidium bromide gradients were prepared bydissolving 22 g of CsCl in 23.7 ml of cleared lysate. 1.125 ml ofaqueous ethidium bromide (10 mg/ml) were added to the solution. Themixture was then centrifuged in polyallomer tubes in a Beckman 60 Tirotor at 44000 rpm for 48-72 hours. The resulting bands of DNA in thegradients were visualized with ultraviolet light and the lower band(covalently closed plasmid DNA) was removed by puncturing the tube withan 18 gauge needle attached to a syringe. Ethidium bromide was removedfrom the plasmid by repeated extraction with 2 volumes ofchloroform-isoamyl alcohol (24:1). Plasmids were then dialyzedextensively against 10 mM Tris-HCl (pH 7.4) containing 0.1 mM EDTA toremove CsCl. The plasmid DNA was then concentrated by ethanolprecipitation.

EXAMPLE V Construction of pBR 322/Vaccinia/Herpes Virus TK RecombinantPlasmids.

FIGS. 3B and 3C summarize the steps involved in the construction of therecombinant plasmids used for inserting the Bam HSV TK fragment into S-or L-variant vaccinia. Approximately 15 μg of covalently closed pDP 3were cleaved by partial digestion with Bam HI (Bethesda ResearchLaboratories) by incubation in Bam HI buffer, consisting of 20 mMTris-HCl (pH 8.0), 7 mM MgCl₂, 100 mM NaCl, and 2 mMbeta-mercaptoethanol, using 7 units of Bam HI for 10 minutes at 37° C.Since pBR 322 and vaccinia Hind III F each contain a Bam HI site,partial cleavage results in a mixture of linear plasmids cut either atthe pBR 322 or vaccinia Bam HI site. These mixed linear plasmids werethen separated from the fragments of pDP 3 cut at both the pBR 322 andvaccinia Bam HI sites by electrophoresis on agarose gels and the singlycut linear plasmids were isolated using glass powder as described inExample I.

A recombinant pBR 322 containing the 2.3 megadalton (md) HSV Bam HIfragment which codes for HSV TK, as described by Colbere-Garapin et al.,Proc. Natl. Acad. Sci. USA 76, 3755-3759 (1979), was digested tocompletion with Bam HI and the 2.3 md Bam TK fragment was isolated froman agarose gel as described above.

pDP 3 Bam TK recombinant plasmids were constructed by ligatingapproximately 1 μg of Bam HI linearized pDP 3 to approximately 0.2 ug ofisolated Bam TK fragment in 20 μl of ligation buffer containing 100units of T4 DNA ligase at 10° C. overnight. This ligation mixture wasthen used to transform competent E. coli HB 101 cells as described inExample III.

EXAMPLE VI Screening of Transformed Cells for Identification of ThoseContaining Recombinant Plasmids Having HSV TK Inserts

Transformed cells containing recombinant plasmids were screened for HSVTK insertions by colony hybridization essentially as described byHanahan et al., Gene 10, 63-67 (1980).

A first set of nitrocellulose filters (Schleicher and Schull BA85) wereplaced on Petri dishes filled with LB agar containing 100 μg/ml ofampicillin. Transformed cells were spread on the filters and the disheswere incubated at 30° C. overnight or until the colonies were justvisible. A replica nitrocellulose filter of each of the first set offilters was made by placing a sterile nitrocellulose filter on top ofeach of the above-mentioned original filters and pressing the twofilters together firmly. Each pair of filters was then notched (keyed)with a sterile scalpel blade, separated, and each filter was transferredto a fresh LB agar plate containing ampicillin at 100 μg/ml for 4-6hours. The first set of filters (original filters) were then placed onLB agar plates containing 200 μg/ml of chloramphenicol to amplifyplasmid production. The replica filters were stored at 4° C.

After 24 hours on chloramphenicol, the original nitrocellulose filterswere prepared for hybridization as follows. Each nitrocellulose filterwas placed on a sheet of Whatman filter paper saturated with 0.5N NaOHfor 5 minutes, blotted on dry filter paper for 3 minutes, and placedback on the NaOH saturated filter paper for 5 minutes to lyse thebacteria thereon and to denature their DNA. This sequence was thenrepeated using Whatman filter paper sheets saturated with 1.0M Tris-HCl(pH 8.0) and repeated a third time with filter paper sheets saturatedwith 1.0M Tris-HCl (pH 8.0) containing 1.5M NaCl for purposes ofneutralization. The nitrocellulose filters treated in this manner werethen air dried and baked in vacuo at 80° C. for 2 hours.

Prior to hybridization these nitrocellulose filters were next treatedfor 6-18 hours by incubating at 60° C. in a prehybridization bufferwhich is an aqueous mixture of 6×SSC [1×SSC=0.15M NaCl and 0.015M Nacitrate (pH 7.2)], 1×Denharts [1×Denharts=a solution containing 0.2%each of Ficoll, BSA, and polyvinylpyrrolidone], and 100-200 μg ofdenaturated sheared salmon sperm DNA (S.S. DNA)/ml, 1 mM EDTA, and 0.1%SDS. This treatment will decrease the amount of binding between thefilter and non-hybridized probe DNA next to be applied to the filters.

To screen for recombinant plasmids containing HSV TK inserts, thetransformed colonies fixed to the original, treated, nitrocellulosefilters were hybridized with ³² P labelled Bam HSV TK fragment byimmersion of the filters in hybridization buffer containing 2×SSC (pH7.2), 1×Denhart's solution, 50 μg of S.S. DNA/ml, 1 mM EDTA, 0.1% ofSDS, 10% of dextran sulfate, and ³² P Bam TK as the hybridization probe.The level of radioactivity of the solution was approximately 100,000counts per minute (cpm) per milliliter.

Hybridization was effected at 60° C. over 18-24 hours [Wahl et al. Proc.Natl. Acad. Sci. USA 76, 3683-3687 (1979)].

[To prepare the hybridization probe, the 2.3 md Bam TK fragment waslabelled by nick translation according to the method of Rigby et al., J.Mol. Biol. 113, 237-251 (1977). More specifically, 0.1 ml of a reactionmixture was prepared containing 50 mM Tris-HCl (pH 7.6), 5 mM MgCl₂, 20μM deoxycytidine triphosphate (dCTP), 20 μM deoxyadenosine triphosphate(dATP), 20 μM deoxyguanosine triphosphate (dGTP), 2 μM (alpha-³²P)deoxythymidine triphosphate (dTTP) (410 Curies/m mol) (AmershamCorporation), 1 ng of DNase I, 100 units of DNA polymerase I (BoehringerMannheim), and 1 ug of Bam TK fragment. The reaction mixture wasincubated at 14° C. for 2 hours. The reaction was terminated by adding50 μl of 0.5M EDTA and heating to 65° C. for 10 minutes. Unincorporatedtriphosphates were removed by gel filtration of the reaction mixture onSephadex G50.]

After hybridization, excess probe was removed from the nitrocellulosefilters by washing 5 times in 2×SSC (pH 7.2) containing 0.1% of SDS atroom temperature, followed by 3 washes in 0.2×SSC (pH 7.2) containing0.1% of SDS at 60° C., with each wash lasting 30 minutes. The washedfilters were then air dried and used to expose X-ray film (Kodak X-omatR) at -70° C. for 6-18 hours using a Cronex Lightening Plus intensifyingscreen (du Pont) for enhancement.

The exposed and developed X-ray film was then used to determine whichcolonies contained pBR 322 vaccinia Bam HSV TK recombinant plasmids.Those colonies which exposed the X-ray film were located on thecorresponding replica nitrocellulose filter. Such positive colonies werethen picked from the replica filters for further analysis. Of theapproximately 1000 colonies screened in this manner, 65 colonies weretentatively identified as having a Bam TK insert within pDP 3.

EXAMPLE VII Restriction Analysis of Recombinant Plasmids Containing BamHSV TK

Each of the 65 colonies which were tentatively identified as containingrecombinant plasmids with Bam HSV TK inserts were used to inoculate 2.0ml cultures of LB broth containing ampicillin at 100 μg/ml. The cultureswere then incubated at 37° C. overnight. Plasmids were extracted fromeach culture as described in Example III. The plasmids were dissolved ina 50 μl of water after isopropanol precipitation.

To determine if the plasmids contained an intact 2.3 md Bam HSV TKfragment and at which Bam HI site within pDP 3 the Bam HSV TK wasinserted, 25 μ1 of each plasmid preparation were mixed with 25 μl of2×Hind III buffer and digested at 37° C. for 2 hours with 1 unit of HindIII. The resulting fragments were then analyzed by electrophoresis on a1.0% agarose gel as described previously.

Of the 65 plasmid preparations analyzed, 6 were found to contain Bam HSVTK fragments inserted into the Bam HI site present in the vaccinia HindIII F portion of the plasmid, i.e. they yielded Hind III restrictionfragments of molecular weights corresponding to linear pBR 322 (2.8 md)and fragments of a molecular weight greater than that of the vacciniaHind III F fragment (8.6 md).

These 6 plasmids were further analyzed with Sst I (an isoschizomer ofSac 1) to determine the number and orientation of the Bam HSV TKfragments inserted within vaccinia Hind III F Fragment, since Sst I (SacI) cleaves both the Bam HSV TK fragment and the vaccinia Hind III Ffragment asymetrically. The analyses were performed by mixing 25 μl ofthe plasmid with 25 μl of 2×Sst buffer [50 mM Tris-HCl (pH 8.0), 10 mMof MgCl₂, 100 mM of NaCl, and 10 mM of DTT] and digesting with 1 unit ofSst I (Bethesda Research Laboratories') at 37° C. for 2 hours. Theresulting fragments were analyzed by electrophoresis in 1% agarose gels.Of the 6 plasmids analyzed, 5 yielded two Sst I fragments with molecularweights of 10.1 md and 3.5 md, indicating a single Bam HSV TK insert.One of these plasmids was selected for further study and designated pDP132. The other plasmid yielded three Sst I fragments with molecularweights of 10.8 md, 2.8 md, and 2.3 md, indicating tandom Bam HSV TKinserts oriented head to tail and in the opposite orientation ascompared to pDP 132. This plasmid was designated pDP 137. The plasmidspDP 132 and pDP 137 are diagramed in FIG. 3C.

EXAMPLE VIII Isolation of a TK⁻ S-variant Vaccinia Virus

To isolate a TK⁻ S-variant vaccinia virus mutant, a virus population wassubjected to strong selective pressure for such a mutant by growing thevirus in cells in the presence of BUdR, which is lethal to organismscarrying the TK gene. More in particular, confluent monolayers of TK⁻human (line 143) cells growing in Eagle's Special medium in 150 mm Petridishes were infected with approximately 3×10³ plaque forming units (pfu)of S-variant vaccinia virus per dish (20 dishes used) in the presence of20 μg BUdR/ml. (Eagle's Special medium is a commercially availablenutrient medium for the growth of most cell lines. Alternative mediasuch as Eagle's Minimum Essential Medium, Basal Eagle's Medium,Ham's-F10, Medium 199, RPMI-1640, etc., could also be used.) Growth isat 37° C. in an atmosphere enriched in CO₂. This is conveniently doneusing a CO₂ -incubator providing air enriched with CO₂ to have a CO₂content of about 5 percent.

Ninety-three of the plaques which developed were isolated and replaquedon TK⁻ human (line 143) cells under the conditions mentioned previouslyand again in the presence of 20 μg of BUdR/ml. A number (5) of large,well isolated plaques were picked for further analysis.

The five plaque isolates were tested for growth on cell monolayers underthe same conditions used earlier and in the presence or absence of 20 μgBUdR/ml. The relative growth of each plaque in the presence and absenceof BUdR was noted and compared with the relative growth in similarmonolayer cell cultures of the parent S-variant virus. The followingresults were obtained:

    ______________________________________                                        Plaque Isolate                                                                             - BUdR (pfu/ml)                                                                           + BUdR (pfu/ml)                                      ______________________________________                                        #70          5.1 × 10.sup.5                                                                      4.1 × 10.sup.5                                   #73 1.0 × 10.sup.6 1.0 × 10.sup.6                                 #76 4.7 × 10.sup.5 4.7 × 10.sup.5                                 #79 5.4 × 10.sup.5 4.4 × 10.sup.5                                 #89 5.9 × 10.sup.5 7.0 × 10.sup.5                                 S-variant  1.7 × 10.sup.10 9.7 × 10.sup.6                       ______________________________________                                    

The growth of plaque isolate #79 was further monitored in the presenceof 0, 20 and 40 μg BUdR/ml and compared with the growth of its parentS-variant virus. The following results were obtained:

    ______________________________________                                        Yield (pfu/ml)                                                                     Virus    0 μg/ml   20 μg/ml                                                                          40 μg/ml                                 ______________________________________                                        #79       2.5 × 10.sup.5                                                                       4.1 × 10.sup.5                                                                   3.2 × 10.sup.5                            S-Variant 1.2 × 10.sup.9 1.3 × 10.sup.6 2.0 ×                                             10.sup.5                                      ______________________________________                                    

In addition, the above 5 plaque isolates and the S-variant parent weremonitored for growth on TK⁻ human (line 143) cells in the presence ofMTAGG. MTAGG is an Eagle's Special medium modified by the presence of:

    ______________________________________                                        8 × 10.sup.-7 M    methotrexate                                           1.6 × 10.sup.-5 M  thymidine                                            5 × 10.sup.-5 M  adenosine                                              5 × 10.sup.-5 M  guanosine                                              1 × 10.sup.-4 M  glycine                                              ______________________________________                                    

(cf. Davis et al., op. cit.) and selects for thymidine kinase andagainst organisms free of the thymidine kinase gene. The results of suchan experiment were as follows:

    ______________________________________                                                       Plaque Forming Units/ml                                        Virus         - MTAGG   + MTAGG                                               ______________________________________                                        #70           4.0 × 10.sup.5                                                                    0                                                       #73 5.8 × 10.sup.5 0                                                    #76 2.8 × 10.sup.5 3.3 × 10.sup.3                                 #79 3.6 × 10.sup.5 0                                                    #80 4.3 × 10.sup.5 4.0 × 10.sup.3                                 S-Variant 4.8 × 10.sup.9 2.6 × 10.sup.9                         ______________________________________                                    

Of the three plaque isolates showing complete inhibition of growth inthe presence of MTAGG, isolate #79 was arbitrarily selected and extractsprepared from cells infected with #79 virus were compared with extractsprepared from uninfected cells and from cells infected with theS-variant parent virus with respect to the ability of the extracts tophosphorylate tritiated (³ H) thymidine. The results are tabulatedbelow:

    ______________________________________                                                         .sup.3 H Thymidine                                              Phosphorylated                                                               Extract Source (cpm/15 μg Protein)                                       ______________________________________                                        Uninfected TK.sup.-- human                                                      (line 143)                                                                    #79 infected cells 90                                                         S-variant infected cells 66,792                                             ______________________________________                                    

In view of (1) resistance to BUdR, (2) inhibition of growth by a mediumcontaining MTAGG, and (3) failure to detect significant phosphorylationof thymidine in infected cell extracts, plaque isolate #79 is consideredto lack thymidine kinase activity. The isolate is designated VTK⁻ 79.

EXAMPLE IX Marker Rescue of L-variant Vaccinia DNA by the S-Variant

Four preparations of L-variant DNA were prepared for marker rescuestudies. The first consisted of purified, intact, L-variant vacciniaDNA. The second consisted of L-variant vaccinia DNA digested with Bst EII, a restriction endonuclease which generates a donor DNA fragment,fragment C, comprising that DNA which is absent from the S-variant anduniquely present in the L-variant and which also has, at both ends ofthe DNA chain, a region of DNA homologous with corresponding sequencesin the S-variant. The third and fourth preparations respectivelyconsisted of L-variant DNA digested with Ava I and Hind III, restrictionendonucleases that cleave the vaccinia genome within the uniqueL-variant DNA sequence. The marker rescue studies performed with thesefour preparations demonstrate that those L-variant DNA fragmentscontaining the deleted region absent from the S-variant can bereintroduced into the S-variant by an in vivo recombination techniqueproviding that the fragment contains, in addition to the deleted region,terminal regions which are homologous with corresponding sequences inthe S-variant.

A better understanding of the fragments employed in these studies willbe had by referring to FIGS. 6A-C, each of which is a restriction map ofa portion of the left terminus of the vaccinia genome. More inparticular, each map refers to the left-terminal region of the genomecomprising approximately 60 kilobasepairs, as indicated in the Figure.The portion of the vaccinia genome which is deleted from the S-variantis represented in each map as the region between the dotted lines shownin the Figures, a region approximately 10 kilobasepairs in length.

Turning now more specifically to FIG. 6A, it is evident that fragment Hobtained by digestion with Ava I is completely within the deleted regionbut will have no terminal DNA fragments homologous with the DNA of theS-variant because the Ava I cleavage sites fall entirely within thedeleted region of the S-variant.

The restriction map of FIG. 6B pertaining to Hind III shows that thisrestriction enzyme similarly fails to produce a L-variant DNA fragmentoverlapping the deleted region of the S-variant. In this instance,sequences homologous with the S-variant are found at the left terminusof the C-fragment of Hind III. However, the restriction site at theright-hand terminus of fragment C falls within the deleted region andthere is no terminal sequence homologous with the DNA sequence of theS-variant.

In contrast, the restriction map shown in FIG. 6C pertaining to Bst E IIshows that digestion with this enzyme produces a fragment, fragment C,which includes the deleted region absent from the S-variant and also hasterminal portions at both the left and right ends which are homologouswith corresponding portions of the S-variant.

The results of the experiments, discussed more in detail below, indicatethat the DNA which is present in the L-variant but is deleted from theS-variant is rescued by the S-variant with high efficiency from theintact L-variant genome, is rescued with lower efficiency from the Cfragment of Bst E II, and cannot be rescued from either of the L-variantDNA fragments prepared with the Ava I and Hind III restrictionendonucleases.

The high efficiency with which the deleted sequence is rescued from theintact L-variant is attributable to the fact that a single crossoverbetween the intact L-variant and the S-variant is sufficient to producean L-variant genome type. On the other hand, to rescue the deletedportion from the C fragment of Bst E II, a crossover between thefragment and the S-variant is necessary in both the left- and right-handterminal portions of the C-fragment in order to incorporate the deletedregion into the S-variant. Finally, since neither digestion with Ava Inor with Hind III produces DNA fragments which can be incorporated intothe S-variant by any crossover, no rescue of the deleted portion iseffected.

The marker rescue was performed on CV-1 monolayers using the calciumphosphate technique of Graham et al., Virology, 52, 456-467 (1973), asmodified by Stow et al. and Wigler et al., both mentioned earlierherein. Confluent CV-1 monolayers were infected with S-variant vacciniavirus to give approximately 50 to 200 plaques in each of a number (5-20)of Petri-dishes of 6 cm diameter. To infect the cells, the growth medium(e.g. Eagle's Special containing 10% calf serum) is aspirated and adilution of the virus containing 50-200 pfu/10.2 ml in a cell-compatiblemedium such as Eagle's Special containing 2% calf serum is applied tothe cell monolayer. After incubation for a period of one hour at 37° C.in a CO₂ -incubator to permit the absorption of the virus to the cells,various of the four L-variant DNA preparations earlier mentioned wereeach separately added to the monolayers as a calcium phosphateprecipitate containing one microgram per dish of the L-variant DNApreparation. After 40 minutes, Eagle's Special medium with 10% calfserum was added and, four hours after the initial addition of the DNA,the cell monolayer was exposed to 1 ml of buffered 25 percent dimethylsulfoxide for four minutes. This buffer contains 8 g of NaCl, 0.37 g ofKCl, 0.125 g of Na₂ HPO₄.2H₂ O, 1 g of dextrose, and 5 g ofN-(2-hydroxyethyl)piperazine,N'-(2-ethanesulfonic acid) (Hepes) perliter, having a final pH of 7.05. The dimethylsulfoxide was removed andthe monolayers were washed and overlayed with nutrient agar. After threedays, at 37° C. in a CO₂ -incubator, the cells were stained with anutrient agar overlayer containing Neutral red dye, which stains theuninfected cells (nutrient agar=Eagle's Special medium containing 10%calf serum and 1% agar). The next day, the agar overlay was removed andthe monolayers were transferred to nitrocellulose filters and preparedfor in situ hybridization as described by Villarreal et al., loc. cit.Since digestion of the L-variant genome with Ava I generates a 6.8kilobasepair fragment, fragment H, that resides entirely with the uniqueDNA sequences deleted in the S-variant genome (cf. FIG. 6A), ³²P-labelled'nick-translated Ava I H fragment provides a highly specificprobe for detecting the rescue of the unique L-variant DNA sequence bythe S-variant.

For hybridization, the nitrocellulose filters were interleaved withWhatman No. 1 filter paper circles in 6 cm Petri dishes and wereprehybridized for 6 hours at 60° C. in prehybridization buffer (SSC,Denhardt solution, EDTA, and S.S. DNA) as described earlier herein inExample VI. The radioactive probe consisting of ³² P-labellednick-translated L-variant Ava I, H fragment, having a specific activityof approximately 1×10⁸ cpm/μg was used for hybridization in 2×SSC,1×Denhardt, 1 mM EDTA, 0.1 percent SDS, 10 percent of dextran sulfate,and 50 μg/ml of sonicated S.S. DNA at approximately 1×10⁵ cpm/mlovernight at 60° C. The radioactive probe was prepared according to themethod of Rigby et al., J. Mol. Biol. 113, 237-251 (1977). The filterswere washed repeatedly at room temperature and at 60° C. using thewashing procedure of Example VI, were air dried, and radioautographed.

The results of the experiments are summarized in Table I below:

                  TABLE I                                                         ______________________________________                                                       Percent of Plagues                                               Donor L-variant Containing L-variant                                          DNA Preparation Genotype                                                    ______________________________________                                        Intact L-variant                                                                             5                                                                Bst E II total digest 0.1                                                     Ava I total digest 0                                                          Hind III total digest 0                                                     ______________________________________                                    

A minimum of 5000 plaques were analyzed for each donor DNA preparation.

EXAMPLE 10 In Vivo Recombination Using pDP 132 and pDP 137 to GenerateVaccinia Virus Mutants vp-1 through vp-6 and Identification ThereofUsing Replica Filters

A first calcium orthophosphate precipitate of donor DNA was prepared bycombining 5 μg of pDP 132 Hind III digested DNA in 50 μg of water, 4 μgof S-variant carrier DNA (prepared as in Example I) in 40 μl of water,and 10 μl of 2.5M CaCl₂, combining the resultant mixture with an equalvolume of 2×Hepes phosphate buffer comprising 280 mM NaCl, 50 mM Hepes,and 1.5 mM sodium phosphate (pH=7.1), and permitting the precipitate toform over a period of 30 minutes at room temperature. A secondprecipitate was prepared in the same fashion, in the same amounts, butusing pDP 137 Hind III digested DNA.

[As described more in detail by Stow et al. loc. cit. and Wigler et al.,loc. cit., the modifications of the Graham et al. precipitationtechnique referred to earlier employ carrier DNA as a high molecularweight substance increasing the efficiency of calcium orthophosphateprecipitate formation. The carrier DNA employed is DNA from the viruswhich is used for infection of the monolayered cells in the in vivorecombination technique.]

For in vivo recombination, confluent monolayers of CV-1 growing inEagle's Special medium containing 10% calf serum were infected withS-variant vaccinia virus at a multiplicity of infection of 1 pfu/cell.The infection procedure is like that described in Example IX. The viruswas permitted to absorb for 60 minutes at 37° C. in a CO₂ -incubator,after which the innoculum was aspirated and the cell monolayer waswashed. The precipitated DNA preparations were applied to separate cellmonolayers and, after 40 minutes at 37° C. in a CO₂ -incubator, liquidoverlay medium was added (Eagle's Special containing 10% calf serum). Ineach case, the virus was harvested after 24 hours at 37° C. in a CO₂-incubator by 3 freeze/thaw cycles and titered on CV-1 monolayers.Approximately 15000 plaques were analyzed on CV-1 monolayers forrecombinant virus using replica filters prepared as follows.

Plaques formed on confluent CV-1 monolayers under a nutrient agaroverlay were transferred to a nitrocellulose filter by removing the agaroverlay cleanly with a scalpel and placing the nitrocellulose filteronto the monolayer. Good contact between the filter and monolayer waseffected by placing a Whatman No. 3 filter paper, wetted in 50 mM Trisbuffer (pH=7.4) and 0.015 mM NaCl over the nitrocellulose filter andtamping with a rubber stopper until the monolayer transferred to thenitrocellulose shows a uniform color surrounding discrete uncoloredplaques. (The monolayer has been previously stained with Neutral red,which is taken up by viable cells, i.e. cells unlysed by virusinfection).

The nitrocellulose filter having the transferred monolayer thereon isnow removed from the Petri dish and placed with the monolayer side up. Asecond nitrocellulose filter, wetted in the above-mentioned Tris-NaClsolution, is now placed directly over the first nitrocellulose filterand the two filters are brought firmly into contact by tamping with arubber stopper after protecting the filters with a dry Whatman No. 3circle. After removing the filter paper, the nitrocellulose filters arenotched for orientation and separated. The second (replica)nitrocellulose filter now contains a mirror image of the cell monolayertransferred to the first nitrocellulose filter. The second filter isconveniently placed in a clean Petri dish and frozen. The firstnitrocellulose filter is subjected to hybridization employing ³²P-labelled Bam HSV TK fragment as a probe. The preparation of the probeand the hybridization technique are described earlier herein in ExampleVI.

Approximately 0.5 percent of the plaques analyzed by hybridization werepositive, i.e. were recombinant virus containing Bam HSV TK.

Plaques of recombinant virus corresponding to those identified on thefirst nitrocellulose filter by hybridization were then isolated from thenitrocellulose replica filter by the following technique for furtherpurification.

Using a sharp cork borer having a diameter slightly larger than theplaque to be picked, a desired plaque is punched out from the first ororiginal nitrocellulose filter which has been used for identification ofrecombinants by hybridization. The resulting perforated filter is nextused as stencil to identify and isolate the corresponding plaque on thereplica filter. Namely, the replica filter is placed with the monolayerside up on a sterile surface and covered with a sheet of Saran wrap. Theperforated first or original nitrocellulose filter is then placedmonolayer side down over the second filter and the orientation notchespresent in the filters are aligned to bring the mirror images of theplaques into register. Again, using a cork borer, a plug is removed fromthe replica filter and, after removal of the Saran wrap protectivelayer, is placed in one ml of Eagle's Special medium containing 2% calfserum. The nitrocellulose plug is sonicated in this medium for 30seconds on ice to release the virus. 0.2 ml of this virus preparation,and 0.2 ml of a 1:10 dilution of the preparation, are plated on CV-1monolayers present in 6 cm Petri dishes.

As a plaque purification step, the entire sequence of preparing a firstnitrocellulose filter, a replica filter, hybridization, and plaqueisolation from the replica filter was repeated.

One sample of a purified plaque prepared in this manner starting from acalcium orthophosphate precipitate of pDP 132 Hind III digested DNA wasdenominated vaccinia virus VP-1. Similarly, a plaque containing arecombinant prepared from pDP-137 Hind III digested DNA was denominatedVP-2. Both samples were grown up on suitable cell cultures for furtherstudy, including identification by restriction analysis and othertechniques.

In like fashion, two further vaccinia mutants respectively denominatedVP-3 and VP-4 were prepared by in vivo recombination employing VTK⁻ 79(an S-variant TK⁻ vaccinia virus as described in Example VIII) as therescuing virus and, respectively, pDP 132 and pDP 137 as the plasmiddonor DNA. The precipitates were formed as described earlier hereinexcept that 5 μg of plasmid donor DNA present in 50 μl of water, 4 μg ofVTK⁻ 79 carrier DNA in 150 μl of water, and 50 μl of 2.5M CaCl₂ werecombined and added dropwise to an equal volume of 250 μl of the Hepesphosphate buffer earlier described.

Further, the cells employed for infection by the VTK⁻ 79 virus carrierwere BHK-21 (Clone 13) cells instead of CV-1.

Two further vaccinia virus mutants denominated VP-5 and VP-6 wereprepared using calcium orthophosphate precipitates of pDP 132 and pDP137, respectively, each as prepared for mutants VP-3 and VP-4. However,in the case of mutants VP-5 and VP-6, the carrier DNA is vaccinia virusVTK⁻ 11, rather than VTK⁻ 79.

Again, BHK-21 (C-13) cell monolayers were infected, the rescuing virusin this case being VTK⁻ 11.

EXAMPLE XI Expression of HSV TK by Vaccinia Mutant VP-2 and the Use ofIDC* for Identification Thereof

The virus product obtained in Example X by the in vivo recombination ofS-variant vaccinia virus and the calcium orthophosphate precipitate ofpDP-137 Hind III digested DNA was plated out on confluent monolayers ofCV-1 cells present on approximately twenty 6 cm Petri dishes at aconcentration giving approximately 150 plaques per dish. The plaqueswere covered with a liquid overlay medium, e.g. Eagle's Special mediumcontaining 10% calf serum. After 24 to 48 hours of incubation at 37° C.in a CO₂ -incubator, the liquid overlay medium was removed from thedishes and replenished in each case with 1.5 ml of the same liquidoverlay medium containing 1-10 μCi of ¹²⁵ I iododeoxycytidine (IDC*).The plates were then further incubated overnight, at 37° C. in anenriched CO₂ atmosphere, after which the cell monolayer present thereonwas stained by the addition of Neutral red to visualize the plaques bycontrast.

The medium was then removed by aspiration, the monolayers were washedthree times with phosphate-buffered saline solution, and the cellmonolayer on each of the plates was imprinted onto a correspondingnitrocellulose filter. The latter was exposed to X-ray film for from 1to 3 days and then developed.

Those viral plaques containing and expressing the HSV TK gene willphosphorylate IDC* and incorporate it into their DNA, rendering the DNAinsoluble. Other, unphosphorylated and unincorporated, IDC* was removedby washing, so that plaques darkening the X-ray film are thoseexpressing recombinant HSV TK gene. Neither CV-1 cells nor vaccinia,although containing TK, will phosphorylate and incorporate IDC* in theselective fashion characteristic of the HSV TK.

After the recombinant organisms has been identified by radioautography,filter plugs were cut from the nitrocellulose filter, placed in 1 ml ofoverlay medium, (Eagle's Special, 10% calf serum), sonicated, andreplated on CV-1 monolayers. The IDC* assay was then repeated further topurify the viral isolates. In this manner, a virus identical to the VP-2mutant identified by hybridization in Example X was isolated by atechnique dependent on the expression of the HSV TK gene presenttherein.

Again, the results of this Example demonstrate the expression of the HSVTK gene, present in the recombinant organisms according to the presentinvention, by certain of those organisms.

Those vaccinia mutants derived from pDP 137, namely VP-2, VP-4, andVP-6, all will express the HSV TK gene present therein byphosphorylation and incorporation of IDC* in the manner described above.However, the variants VP-1, VP-3, and VP-5, derived from pDP 132, willnot so express the gene, possibly because the orientation of the genewithin the virus is contrary to the direction of gene transcription.

EXAMPLE XII The use of a Selective Medium for the Identification andIsolation of Recombinant Virus Containing HSV TK Gene

Viruses prepared according to Example X by the in vivo recombination, inBHK-21 (C-13) cells, of VTK⁻ 79 vaccinia virus and a calciumorthophosphate precipitate of pDP 137 were used to infect human (line143) TK⁻ cells. More in particular, cell monolayers, in five Petridishes 6 cm in diameter, were each infected with the virus of Example Xat a dilution of the virus from 10⁰ to 10⁻⁴ in the presence of selectiveMTAGG medium. The infection technique was as described earlier.

Five well-separated plaques were isolated and one was replated on CV-1monolayers for a second cycle of plaque purification. One furtherwell-separated plaque, purified twice by plaque purification, was chosenand analyzed. A well-isolated plaque, thus twice plaque-purified, wasselected and analyzed for the presence of the HSV TK gene by in situhybridization employing ³² P-labelled Bam HSV TK. The hybridizationtechnique was, again, as described earlier. The mutant vaccinia virus,positive for the presence of the HSV TK gene, was denominated VP-4.

EXAMPLE XIII Construction of pDP 120

About 20 micrograms of pDP 3 were digested with Pst I and the fragmentsobtained were separated on an agarose gel in a procedure analogous tothat discussed in detail in Example I. The Pst I fragment having amolecular weight of 3.7 md, corresponding to the middle portion of thevaccinia Hind III F-fragment, was isolated.

Approximately 500 nanograms of this fragment were then ligated with 250ng of pBR 322, previously cleaved with Pst I, in 20 microliters ofO'Farrell buffer (OFB) [cf. O'Farrell et al., Molec. Gen. Genetics 179,421-435 (1980)]. The buffer comprises 35 mM of tris acetate (pH 7.9), 66mM of potassium acetate, 10 mM of magnesium acetate, 100 μg/ml of bovineserum albumin, and 0.5 mM of dithiothreitol. For purposes of ligation, 1mM of adenosine triphosphate (ATP) and approximately 20 units of T4 DNAligase (New England Biolabs) were present. The mixture was maintained at16° C. for 16 hours.

The ligation mixture was then used to transform competent E. coli HB101. Amp^(S), Tet^(R) recombinants were selected on appropriateantibiotic plates, analogous to the procedure described in Example III.Several recombinant plasmids were then analyzed by restriction analysiswith Pst I and Bam HI, as in Example VII, to confirm their construction.One colony containing a plasmid with the correct construction was grownon a large scale and recovered as in Example IV and was designated pDP120.

EXAMPLE XIV Construction of pDP 301 A and 301 B; Construction of VP 7and VP 8

Approximately 10 micrograms of plasmid pDP 3 were cleaved with Hind IIIand the 8.6 md vaccinia F-fragment was isolated in an amount ofapproximately 5 micrograms from an agarose gel using a techniqueanalogous to that discussed in Example I. The fragment was self-ligatedby incubating for 16 hours at 16° C. in 1.0 ml of O'Farrell buffer (OFB)containing 1 mM ATP and 80 units of T4 DNA ligase.

After incubation, the reaction was terminated by heating at 65° C. forten minutes and the DNA was then precipitated with ethanol.

The self-ligated Hind III F-fragment was then resuspended in 250 μl ofOFB and digested for four hours with 30 units of Bam HI. The reactionwas terminated by heating at 65° C. for ten minutes and the resultantDNA fragments were separated on a one percent agarose gel. The bandcorresponding to the 8.6 md Hind III F-fragment, which fragment had beeninverted around the Bam HI site, was then isolated using techniquespreviously described.

This inverted Hind III F-fragment was then inserted into the Bam HI siteof pBR 322 as follows.

pBR 322 was cleaved with Bam HI by conventional techniques. The linearplasmid was then treated with calf intestine alkaline phosphatase (CIAP)to remove the 5'-phosphates, thus discouraging re-circularization[Chaconas et al., Methods in Enzymol. 65, 75 (1980)]. More inparticular, 5 μg of linear pBR 322 in 400 μl of OFB, adjusted to pH 9.0,were combined with 0.75 units of CIAP (Boehringer Mannheim) for 30minutes at 37° C. A further 0.75 unit of CIAP was added and the mixturedigested for 30 minutes at 60° C. The DNA was then deproteinized byphenol extraction as described in Example I for the purification ofvaccinia DNA.

About 450 ng of the pBR 322 DNA treated as above were then ligated toabout 400 ng of inverted Hind III F-fragment in 15 μl of OFB containing1 mM ATP and 20 units of T4 DNA ligase at 16° C. over a period of 16hours. The ligation mixture was then used directly to transform E. coliHB 101 cells as previously described in Example III. The transformedbacteria were then screened for Amp^(R), Tet^(S) recombinants by platingon appropriate antibiotic plates, again as described in Example III.

The recombinant plasmids were screened by Hind III restriction analysisof minilysates (again as described in Example III) to determine which ofthe plasmids contained an inverted Hind III F-fragment in eitherorientation. Those two plasmids containing the fragment in differentorientations were designated respectively as pDP 301 A and pDP 301 B.

These plasmids were used to construct two new recombinant vacciniaviruses each containing pBR 322 DNA sequences inserted into the Bam HIsite of the vaccinia Hind III F-fragment in opposite orientations.

More in particular, pDP 301 A and pDP 301 B were inserted into VTK⁻ 79by in vivo recombination, as described in Example X. However, 10 μg ofdonor DNA (either pDP 301 A or pDP 301 B, digested with Sst I) and 2 μgof VTK⁻ 79 carrier DNA were used to prepare the calcium ortho- phosphateprecipitate which was employed for addition to CV-1 cells which wereinfected with VTK⁻ 79 as the rescuing virus.

The recombinant viruses were then screened by using the replica filtertechnique also disclosed in Example X using nick-translated pBR 322 DNAas the probe.

The virus in which pBR 322 DNA sequences from pDP 301 A had beenrecombined in vivo with VTK⁻ 79 was designated as VP 7: the recombinantvirus containing pBR 322 DNA from pDP 301 B was designated as VP 8.

EXAMPLE XV Construction of pJZ 102 A/F; Construction of VP 9

A plasmid containing the complete cDNA sequence coding for thehemagglutinin (HA) gene of the influenza virus A/PR/8/34, inserted intopBR 322, is one of the plasmids made by Bacz et al. in Nucleic AcidsResearch 8, 5845-5858 (1980). The plasmid contains the completenucleotide coding sequence for the HA gene inserted at the Hind III siteof pBR 322.

The hemagglutinin sequence in the plasmid was switched in direction bydigesting about 500 ng of the original plasmid, designated pJZ 102 A,with Hind III in OFB, then religating using T4 DNA ligase and ATP aspreviously described.

The ligation mixture was then used to transform competent E. coli andthe bacteria were screened for Amp^(R), Tet^(S) colonies. Recombinantplasmids from minilysis preparations were then screened forhemagglutinin sequences present in opposite orientations by Ava Idigestion of the plasmids and analysis on agarose gels. Those plasmidsin which the HA sequence was present in a direction opposite to thatfound in pJZ 102 A were designated pJZ 102 B (cf. FIG. 9A).

Approximately 500 ng of pJZ 102 A were linearized by digestion with BamHI in OFB as previously described. The linearized pJZ 102 A was ligatedwith approximately 500 ng of inverted Hind III F-fragment, the latterbeing conveniently obtained by Bam HI digestion of pDP 301 A (cf.Example XIV). Ligation took place in 20 μof OFB containing 1 mM of ATPand approximately 20 units of T4 DNA ligase at 16° C. over a period of16 hours.

The ligation mixture was used directly to transform competent E. coli RR1 cells [Bolivar et al, Gene 2, 95-113 (1977)] as previously described.

Transformed cells were plated on ampicillin plates and screening forrecombinants was effected by colony hybridization using nick-translatedvaccinia Hind III F-fragment DNA as the probe, all as previouslydescribed in Example VI.

DNA from colonies found by hybridization to be positive was thenanalyzed by Hind III restriction analysis and agarose gelelectrophoresis.

A plasmid containing pJZ 102 A inserted into the Bam HI site of vacciniaHind III F-fragment was isolated and designated as pJZ 102 A/F.

Using the in vivo recombination technique described in detail in ExampleXV, 10 μg of circular donor DNA from pJZ 102 A/F were used forrecombination, together with 2 μg of VTK⁻ 79 carrier DNA, into VTK⁻ 79vaccinia virus. Recombinant viruses were screened by the replica filtertechnique using a nick-translated Hind III HA fragment as the probe. Therecombinant virus thus isolated was designated as VP 9.

EXAMPLE XVI Construction of VP 10

Plasmid pJZ 102 B (cf. FIG. 10A) was inserted into VP 7 by in vivorecombination using the standard protocol employing 10 μg of circulardonor pJZ 102 B DNA, 2 μg of VTK⁻ 79 carrier DNA, and CV-1 cells.Screening for recombinant viruses containing HA sequences was by thereplica filter technique already described herein, using anick-translated Hind III HA fragment as the probe.

A positive plaque was isolated, plaque purified, and designated as VP10.

EXAMPLE XVII Determination of the Expression of the HA Gene by VP 9 andVP 10

Two 6 cm petri dishes containing BHK-21 cells in a nutrient medium wereinfected with about 200 pfus of A/PR/8/34 influenza virus. Another pairof 6 cm petri dishes containing a monolayer of CV-1 cells in a nutrientmedium were infected with about 200 pfus of VP 9 vaccinia variant, and athird pair of 6 cm petri dishes containing a CV-1 monolayer in anutrient medium were infected with, again, about 200 pfus of VP 10.

The viruses were grown for 48 hours at 37° C. and were stained withNeutral red for one hour at 37° C. to visualize the plaques. Thenutrient medium was then aspirated and the cell monolayers were washedthree times with phosphate buffered saline (PBS) containing 1 mg/ml ofbovine serum albumin (BSA).

1.5 ml of PBS-BSA containing 5 μl of H1 HA rabbit antiserum were nextadded to one of each of the three pairs of petri dishes and the disheswere incubated for one hour at room temperature. A second set of threecell cultures (one BHK and two CV-1 cultures) were treated with 1.5 mlof PBS-BSA containing 5 μl of H3 HA rabbit antiserum and again incubatedfor one hour at room temperature.

Next, all of the cell monolayers were washed three times with PBS-BSAand then 1.5 ml of PBS-BSA containing approximately 1 μCi of ¹²⁵I-labelled protein A (New England Nuclear) was added to each of the 6petri dishes. The dishes were then incubated for approximately 30minutes at room temperature and the radioactive material was aspirated.The cell monolayers were washed five times with PBS-BSA. The cellmonolayer on each of the six plates was then imprinted onto acorresponding nitrocellulose filter and the latter were exposed to X-rayfilm for from one to three days. The film was then developed.

The radioautographs showed complex formation in that petri dish in whichthe BHK cell monolayer had been infected with A/PR/8/34 and treated withH1 HA antiserum. Similarly, exposed film was found for the CV-1 cellmonolayer infected with VP 9 and treated with H1 HA antiserum, alsoindicative of antigen-antibody complex formation for this sample. Allfour other samples were negative for complex formation.

EXAMPLE XVIII Determination of HA Expression by VP 9 in Rabbits

Two New Zealand white rabbits were each infected with 4 OD (at A₂₆₀)units of purified VP 9 mutant vaccinia virus either intravenously (IV)(rabbit No. 1) or intramuscularly (IM) (rabbit No. 2).

Each rabbit was bled and antiserum collected before infection (preimmuneserum) and at 17 days, 25 days, and 41 days after infection. Antiserumfrom each rabbit was tested for its ability to neutralize vaccinia virusinfection as follows.

Serial dilutions of the antisera were prepared in standard virusplaqueing medium (Eagles's special medium containing 2% of fetal bovineserum), then mixed with an equal volume of infectious vaccinia virus(100-300 pfus). Each mixture was held at 4° C. overnight, then used toinfect CV-1 monolayers in 60 mm petri dishes as previously described.Specific vaccinia neutralizing antibodies present in the serum wereindicated by a reduction in the total number of plaques formed. Theresults of such an assay are shown in following Table II.

                  TABLE II                                                        ______________________________________                                        Measurement of Vaccinia Neutralizing Antibodies Induced by                      VP 9 in Rabbits                                                                                  Final Dilution of Antiserum                                Approximate Plaque Indicated Plague Reduction                               Giving           Rabbit No. 1                                                                            Rabbit No. 2                                         Reduction (% of control) (IV) (IM)                                          ______________________________________                                                         17 days                                                      50%              1:64000    1:16000                                             90% 1:10000 1:2000                                                                           25 days                                                      50%               1:128000 1:8000                                               90% 1:32000 1:2000                                                                           41 days                                                      50%               1:128000  1:16000                                             90% 1:32000 1:2000                                                          ______________________________________                                    

As a control, preimmune antiserum at a 1:20 dilution, or no antiserum,gave approximately 180 plaques in the above assay for rabbit No. 1 and240 plaques for rabbit No. 2.

A ¹²⁵ I protein A assay was performed as described in Example XVII. Morein particular, three monolayers of BHK-21 cells infected with A/PR/8/34serum were treated with 25 ul each of either anti-A/PR/8/34 serum,anti-vaccinia serum, or anti-VP 9 serum from the 41-day bleeding ofrabbit No. 1.

Washing and treatment with ¹²⁵ I protein A of each monolayer was asdescribed in Example XVII.

Similarly, three monolayers of cells infected with vaccinia virus(S-variant) were treated with the same three antiserum preparations andalso reacted with ¹²⁵ I protein A as previously described.

The results are presented in following Table III and indicate that VP 9can elicit production, by a rabbit, of antibodies to influenzahemagglutinin expressed by VP 9.

                  TABLE III                                                       ______________________________________                                                      BHK Cells CV-1 Cells Infected                                      Infected with with Vaccinia Virus                                            Antiserum A/PR/8/34 (S-variant)                                             ______________________________________                                        Anti-A/PR/8/34                                                                              +         -                                                       Anti-vaccinia - +                                                             Anti-VP 9 + +                                                               ______________________________________                                    

The production of HA antibodies by a VP 9-infected rabbit was alsotested by measurement of the hemagglutinin inhibition (HI) titer.

Namely, HI tests were performed on the preimmune, 25 day, and 41 dayserum from rabbit No. 1 (IV) according to standard protocol described indetail in "Advanced Laboratory Techniques for Influenza Diagnosis,Immunology Series No. 6, Procedural Guide 1975", U.S. Dept. HEW, PublicHealth Service, Center for Disease Control, Bureau of Laboratories,Atlanta, Ga.

Table IV below shows the HI titers determined using 3+ HA units with thevarious antisera tested. Extracts of BHK cells infected with A/PR/8/34influenza virus were used as a source of HI hemagglutinin.

                  TABLE IV                                                        ______________________________________                                        Hemagglutinin Inhibition                                                           Antiserum                    Titer                                       ______________________________________                                        Anti-A/PR/8/34   greater than 1:320                                             Preimmune serum less than 1:10                                                25 day antiserum  1:60                                                        41 day antiserum greater than 1:320                                         ______________________________________                                    

EXAMPLE XIX Construction of pDO 250 A and 250 B. Construction of VP 11and VP 12

A plasmid pTHBV 1 can be constructed by the methods described byHirschman et al., Proc. Natl. Acad. Sci. USA 77, 5507-5511 (1980),Christman et al., Proc. Natl. Acad. Sci. USA 79, 1815-1819 (1982).

About 20 micrograms of the pTHBV 1 plasmid containing two HBV genomes(subtype ayw) in a head-to-tail arrangement inserted into the Eco R1site of pBR 322 were cleaved with Bgl II restriction endonuclease.

The fragments were separated on a 1.0 percent agarose gel and a 1.5 mdfragment of HBV DNA sequences coding for the HBV surface antigen andpre-surface antigen was isolated (cf. Galibert et al., op. cit.).

About 500 ng of pDP 120 were finally digested with Bam HI underconditions similar to those described in Example V earlier herein.

The resulting digest was then treated with calf intestine alkalinephosphatase (CIAP) in a manner analogous to that described earlierherein in Example XIV. Finally, the aforementioned fragment of BglII-cleaved pTHBV 1 plasmid was ligated into the Bam HI site of theCIAP-treated pDP 120 Bam HI digest under conditions similar to thosedescribed earlier herein.

The ligation mixture was used directly to transform competent E. coli RR1 cells, also as previously described.

The resulting Amp^(S), Tet^(R) colonies were screened for recombinantplasmids by digesting minilysates of possible recombinants with Xho Iand Pst I and analyzing on an agarose gel.

Two recombinant plasmids were isolated, corresponding with insertion,into the Bam HI site present in the vaccinia portion of pDP 120, of theHBV Bgl II fragment in each of two possible directions. The plasmidswere designated as pDP 250 A and pDP 250 B (cf. FIG. 11).

Finally, recombinant vaccinia viruses containing the HBV surface antigenand presurface antigen sequences were constructed by in vivorecombination (see Example X) using 20 μg each of either circular pDP250 A or pDP 250 B and 2 μg of VTK⁻ 79 carrier DNA, with VTK⁻ 79 as theinfecting virus, all as previously described.

The viruses were screened for recombinants using the replica filtertechnique with nick-translated pTHBV DNA as a probe.

The resulting recombinant vaccinia viruses containing the Bgl IIfragments of HBV virus in one of two directions were designated as VP 11and VP 12, as shown in FIGS. 11D and E. (The normal direction of HBVtranscription is indicated for the plasmids in FIG. 11D.)

EXAMPLE XX Construction of pDP 252 Construction of VP 13

20 μg of plasmid pTHBV 1 (cf. Example XIX) were digested with Hha Irestriction endonuclease. The largest fragment of the Hha I digestcomprises 1084 base pairs and contains that entire sequence of thehepatitis B virus coding for the surface antigen, without the regioncoding for the pre-surface antigen (cf. Galibert et al., op. cit.)

This fragment was inserted into pBR 322 at the Hind III site using HindIII linkers. More in particular, approximately 400 ng of HBV Hha Ifragment, isolated from a preparative gel as previously described, weretreated with six units of T4 DNA polymerase (P/L Biochemicals) presentin 40 μl of OFB also containing 2 mM each of deoxyadenosine triphosphate(dATP), deoxyguanidine triphosphate (dGTP), deoxycytosine triphosphate(dCTP), and deoxythymine triphosphate (dTTP). The mixture was incubatedat 37° C. for 30 minutes to trim the extending 3'-ends of the fragment,generated by Hha I restriction endonuclease (cf. O'Farrell et al., op.cit.)

After the reaction period, approximately 500 ng of phosphorylated HindIII linkers (Collaborative Research), 2.5 μl of 20 mM adenosinetriphosphate, 1 μl of 100 mm spermidine (Cal Biochem.), and 1 μl(approximately 80 units) of T4 DNA ligase were added and incubation wascontinued at 10° C. for sixteen hours.

The reaction was stopped by heating at 65° C. for ten minutes and 400 ngof pBR 322 were added.

The Hind III linkers and pBR 322 were then cleaved by addingapproximately 20 units of Hind III and digesting the mixture at 37° C.for four hours.

Once more, the reaction was stopped by heating at 65° C. for ten minutesand the unligated linkers were removed by spermine precipitationaccording to Hoopes et al., Nucleic Acids Research 9, 5493 (1981).

More specifically, 2.5 μl of 0.2M spermine in H₂ O were added to thereaction mixture to make it 10 mM in spermine. The reaction mixture wasincubated on ice for 15 minutes and the precipitate which formed wascollected by centrifugation. Residual spermine was removed from the DNAby resuspending the DNA pellet in 75 percent ethanol, 0.3M sodiumacetate, and 10 mM of magnesium acetate. This mixture was incubated onice for 60 minutes. Residual spermine dissolves in the ethanol, leavinga suspension of DNA which was again pelleted by centrifugation andredissolved in 20 μl of OFB containing 1 mM of ATP and approximately 20units of T4 DNA ligase. Ligation of the pBR 322 and Hind III-linkedfragment was carried out for 16 hours at 10° C.

The ligation mixture was then used directly to transform competent E.coli RR1 cells as previously described. The transformants were platedonto ampicillin plates and screened by colony hybridization aspreviously described herein. A nick-translated HBV Hha I fragment wasused as the probe.

Colonies proved positive by hybridization were analyzed by restrictiondigestion of minilysates. A plasmid containing the Hha I fragment,inserted at the Hind III site, was characterized and designated as pDP252.

A recombinant vaccinia virus containing the Hha I HBV fragment codingfor the HBV surface antigen was constructed using the standard in vivorecombination protocol as set forth in Example XV using 10 μg ofcircular pDP 252 as the donor DNA with 2 μg of VP 8 DNA as the carrierDNA and VP 8 as the infecting virus for CV-1 cells.

The viruses were screened for recombinants using the replica filtertechnique with a nick-translated Hha I HBV fragment as the probe.

The resulting recombinant virus was designated as VP 13.

EXAMPLE XXI Construction of pBL 520 A and 520 B Construction of VP 14and VP 16

Approximately 20 μg of herpes virus type I, strain KOS, DNA, extractedas described by Pignatti et al., Virology 93, 260-264 (1979) weredigested with Eco RI and the resulting fragments were separated on anagarose gel. Eco RI fragment F was isolated from the gel by conventionaltechniques.

About 200 ng of the Eco RI fragment F were ligated with 60 ng of pBR322, digested with Eco RI and subsequently treated with calf intestinealkaline phosphatase in a manner described earlier herein in ExampleXIV. The CIAP treated pBR 322 and the Eco RI F-fragment were ligated in20 μl of OFB containing 1 mM of ATP and approximately 80 units of T4 DNAligase at 16° C. over a period of 16 hours.

The entire ligation mixture was used to transform competent E. coli RR Icells as described in earlier Examples.

The transformed E. coli were grown on ampicillin plates and the Amp^(R),Tet^(R) transformed E. coli were screened for recombinant plasmids byrestriction analysis of minilysates as previously described. Restrictionanalysis was done with Hpa I and Eco RI to determine the orientation ofthe insertion of the Eco RI F-fragment in the plasmid.

The two plasmids thus obtained having the HSV Eco RI F-fragment insertedinto pBR 322 in each of two opposite orientations were designated pBL520 A and pBL 520 B, as shown in FIG. 13B.

Two new vaccinia recombinants, VP 14 and VP 16, were constructed by invivo recombination techniques described in Example X and XV using theseplasmids and VP 7. More specifically, 20 μg each of pBL 250 A or pBL 250B, 2 ug of VP 7 carrier DNA, and VP 7 virus were used to treat CV-1cells to effect the in vivo recombination. Recombinant viruses werescreened by the replica filter technique using nick-translated HSV EcoRI F-fragment as the probe.

EXAMPLE XXII Construction of pBL 522 A and 522 B Construction of VP 17and VP 18

Approximately 20 kg of pBL 520A (cf. Example XXI) were digested with BamHI and the resulting fragments separated on an agarose gel. A 5.1 mdfragment corresponding with the Bam HI G-fragment of HSV DNA (strainKOS) was isolated from the gel using techniques such as those describedin Example I.

Plasmid pDP 120 was partially digested with Bam HI to linearize theplasmid using techniques analogous to those described previously inExample V. The digested plasmid was further treated with calf intestinealkaline phosphatase as in Example XIV to prevent recirculation.

Approximately 100 ng of the pDP 120 DNA so treated were ligated with 120ng of the previously described Bam HI G-fragment in 20 μl of OFBcontaining 1 mM ATP and approximately 80 units of T4 DNA ligase at 160°C. over a period of 16 hours.

Thereafter, the ligation mixture was used directly to transformcompetent E. coli RR I cells as described in previous Examples.

The transformed E. coli were then screened for Amp^(S), Tet^(R)recombinants by colony hybridization as previously described using HSVEco RI fragment as the probe. Colonies positive by hybridization werescreened by restriction analysis of minilysates with Bam HI and Sst I todetermine if the complete HSV Bam HI G-fragment had been inserted and todetermine its orientation within the resulting plasmid.

Two recombinant plasmids were found in this way, each containing the HSVBam HI G-fragment in opposite orientations in the parent plasmid pDP120. The new plasmids were designated pBL 522 A and pBL 522 B.

Again using the in vivo recombination technique described in detail inExamples X and XV herein, 20 ug of donor pBL 522 A or B wererespectively combined with 2 ug of carrier VTK⁻ 79 DNA to form a calciumorthophosphate precipitate. This and the vaccinia virus VTK⁻ 79 wereused to treat CV-1 cells, with the production of two virus mutantsdesignated as VP 17 and VP 18, respectively.

The vaccinia mutants were identified using the relica filter techniquewith HSV Eco RI F-fragment as probe.

EXAMPLE XXIII Construction of an L-variant TK⁻ Vaccinia Virus from theTK⁻ S-variant

In wild-type vaccinia virus, the vaccinia TK gene is known to be presentin the Hind III J-fragment [Hruby et al., J. Virol. 43, 403-409 (1982)].Hence, the Hind III J-fragment of the TK⁻ 79 S-variant vaccinia virus ofExample VIII must have a mutation in the TK gene which inactivates thegene.

A TK⁻ L-variant vaccinia virus war derived in the following manner usingthe Hind III J-fragment of the TK⁻ 79 S-variant.

The Hind III J-fragment of TK⁻ 79 was inserted into pBR 322 in a mannerlike that for the Hind III F-fragment in Example II. The resultingplasmid was used in the standard in vivo recombination protocol,specifically using 10 μg of the plasmid donor DNA, 2 μg of L-variantvaccinia DNA as. carrier, and L-variant vaccinia virus-infected CV-1cells.

Progeny virus was used to infect human TK⁻ cells (line 143) (earlierdescribed) in the presence of 40 μg of BUdR. Virus which grew was plaquepurified in the presence of BUdR and virus from a single plaque waschosen and designated VTK⁻ 79 L (ATCC No. VR 2056). It cannot bedetermined whether the new virus is a spontaneous mutation or is arecombinant containing the J-fragment of the TK⁻ 79 S-variant: thelatter is more likely.

EXAMPLE XXIV Construction of pDP 202

About 34 μg of L-variant vaccinia virus DNA was digested to completionin Ava I buffer [20 mM tris-HCL (pH 7.4), 30 mM Nacl, 10 mM MgCl₂ ] withAva I restriction endonuclease and the resulting fragments wereseparated on an agarose gel as previously described. The Ava IH-fragment was then isolated from the agarose gel.

Approximately 400 ng of pBR 322 in 50 μl of Hind III buffer weredigested to completion with Hind III. Reaction was terminated by heatingat 65° C. for 10 minutes, at which time 45 μl (approximately 600 ng) ofthe isolated vaccinia Ava I H-fragment were added. Then, the totalmixture was precipitated with ethanol. The resulting DNA pellet wasredissolved in 9.5 μl of T4 DNA polymerase buffer [20 mM tris-HCl (pH7.6), 10 mM MgCl₂, 1 mM dithiothreitol, 33 μM dTTP, 33 μM dGTP, 33 μMdCTP, and 33 μM dATP]. The protruding 5'-ends of the DNA fragments werefilled in by adding 1.5 units of T4 DNA polymerase and incubating at 37°C. After 30 minutes, 0.5 μl of a 0.02M solution of ATP was added to makethe reaction mixture 1 mM with respect to ATP, together with 1 μl(approximately 80 units) of T4 DNA ligase. Ligation was then carried outat 10° C. for 20 hours.

The entire ligation mixture was used directly to transform competent E.coli HB 101.

Transformed bacteria were plated on nitrocellulose filters placed onampicillin plates. Recombinant colonies were screened by colonyhybridization using nick-translated vaccinia Ava I H-fragment as aprobe.

Plasmids isolated from colonies which were positive by colonyhybridization were digested with Hind III and analyzed on an agarose gelas previously described. One such plasmid which contained an Ava IH-fragment inserted at the Hind III site of pBR 322 was purified anddesignated pDP 202. Further characterization of this plasmid byrestriction analysis with Sst I and Bam HI determined the orientation ofthe fragment within the plasmid (cf. FIG. 15A).

EXAMPLE XXV Construction of Plasmids pDP 202 TK/A-F; Construction of VP22

Plasmids pDP 202 TK/A-F were constructed by inserting the Bgl II/Bam HITK fragment of HSV into each of the three Bam HI sites in the vacciniaAva I H-fragment portion of pDP 202. The Bgl II/Bam HI fragment containsthe coding region for the HSV TK gene, but not the associated HSVpromoter sequence [McNight et al., Cell 25, 385-398 (1981)].

This was accomplished first by isolating a linear pDP 202 plasmid (7.3md) which had been linearized at a Bam HI site by partial digestion ofthe plasmid with Bam HI. The Bgl II/Bam HI TK fragment was prepared bydigesting the HSV Bam TK plasmid (cf. Example V) with Bgl II and Bam HI.Bgl II digestion cleaves the Bam HI TK fragment at one site, resultingin a 1.8 md fragment containing the coding region of the TK gene and a0.5 md fragment corresponding to the 5'-end of the Bam HI TX fragmentcontaining the HSV promoter. The 1.8 md Bgl II/Bam HI fragment wasisolated from an agarose gel.

To construct the plasmids pDP 202 TK/A-F, approximately 500 ng of Bam HIlinear pDP 202 which had been treated with CIAP as previously describedwas ligated with 250 ng of the aforementioned Bgl II/Bam HI TK fragmentin 20 μl of OFB containing 1 mM ATP and approximately 100 units of T4DNA ligase at 16° C. for 16 hours. The entire ligation mixture was thenused to transform competent E. coli RR I cells as previously described.Transformed cells were plated on ampicillin plates and the colonies werescreened for recombinant plasmids by restriction analysis of minilysateswith Bam HI to determine at which Bam HI site the Bgl II/Bam HI TXfragment was inserted, and in which orientation. The site and directionof orientation were confirmed by restriction analysis with Sst I. Bythis procedure, the Bgl II/Bam HI TX fragment was found to be insertedinto each of the three Bam HI sites in the vaccinia Ava I H-fragment inboth orientations. Each plasmid pDP 202 TK was given a letterdesignation from A to F (cf. FIG. 15 C).

Preparative amounts of plasmids were then grown and purified and used toconstruct recombinant viruses using the standard in vivo recombinationprotocol of Example X and XV. That is, approximately 20 μg of donor DNAfrom each recombinant plasmid were mixed with 2 μg of VTK⁻ 79 DNA as acarrier and were added, in the form of a calcium phosphate precipitate,to a monolayer of CV-1 cells infected with VTK⁻ 79 L virus. Recombinantviruses were screened by using the replica filter technique earlierdescribed using nick-translated HSV Bam TK DNA as a probe.

One recombinant virus which was isolated from in vivo recombination ofVTK⁻ 79 L and pDP 202 TK/E was isolated and designated as VP 22. Thismutant virus was of particular interest because it induced a higherlevel of HSV TK activity in infected cells than does VP 2, VP 4, or VP 6(earlier described herein) as measured by a ¹²⁵ I-iododeoxycytidineassay (IDC).

More in particular, either L-variant vaccinia, VP 4, or VP 22 were usedto infect monolayers of CV-1 cells at the appropriate dilutions to yeild200 plaques per 60 mm Petri dish. Each dish was then treated with ¹²⁵ IIDC and washed as previously described in Example XI to compare thelevels of HSV TK activity.

Infected cell monolayers were then lifted onto nitrocellulose filterswhich were placed on a single sheet of X-ray film to compare the levelsof TK activity by comparing the relative exposure (darkening) of thefilm by each filter.

The results of the IDC assay indicated that L-variant vaccinia containedno HSV TX activity and therefore did not expose the film. VP 4, shownearlier in Example XI to contain HSV TK activity, caused a faintdarkening of the film, VP 22 caused 15-20 times the exposure of VP 4,indicating a significantly higher level of HSV TK activity.

EXAMPLE XXVI Construction of pRW 120

To delete the Bam HI site in pBR 325 (commercially available fromBethesda Research Laboratories), 500 ng of pBR 325 were digested with 8units of Bam HI in 50 μl 1×OFB for 2 hours at 37° C. The reaction wasstopped by heating at 65° C. for 10 min. and the DNA was ethanolprecipitated. The DNA was resuspended in 20 μl 1×OFB containing 100 μMeach of dATP, dGTP, dCTP, and dTTP and 6 units of T4 DNA polymerase andincubated for 30 minutes at 37° C. to fill in the Bam HI sticky ends.The reaction was then stopped by heating at 65° C. for 10 minutes. 1 μlof 0.02M ATP and 0.5 μl (200) units of T4 DNA ligase were added and theDNA was ligated overnight at 16° C. The entire ligation reaction mixturewas used to transform competent E. coli RR 1 cells.

Transformed cells were plated on LB agar plates containingchloramphenicol at 30 μg/ml. Several colonies were then picked and theirplasmid DNA isolated from minilysates. The plasmids were screened fordeletion of the Bam HI site by digestion with Bam HI and analysis onagarose gels. One plasmid resistant to cleavage with Bam HI wasdesignated pBR 325 (Bam X). Preparative amounts of plasmid were thenprepared and purified.

Plasmid pRW 120 was then constructed by inserting the 3.7 md Pst Isubfragment of the Hind III vaccinia F fragment into the Pst I site ofpBR 325 (Bam X). This Pst subfragment was isolated from pDP 120 usingthe glass powder technique previously described.

Approximately 800 ng of the 3.7 md Pst I subfragment were ligated to 400ng of pBR 325 (Bam X), digested with Pst I, and treated with calfintestine alkaline phosphatase in 20 μl 1×OFB containing 1 mM ATP and0.2 μl (80 units) of T4 DNA ligase for 16 hours at 16° C. The entireligation mixture was then used to transform competent E. coli RR1 cells.Aliquots of 10 and 100 μl were then plated on LB agar plates containing30 μg/ml of chloramphenicol.

Plasmids were isolated from Cam^(R) colonies by minilysis and analyzedby digestion with Pst I and Bam HI. One such plasmid containing the PstI fragment inserted into pBR 325 (Bam X) was designated pRW 120.

EXAMPLE XXVII Construction of pDP 122

To clone the A/PR/8-34 HA gene into the Bam HI site of the Hind IIIvaccinia F fragment, it is necessary to change the Hind III ends of theHA sequence found in pJZ 102 to Bam HI sticky ends.

20 μg of pJZ 102 were digested with 20 units of Hind III. (This and allsubsequent restriction endonuclease digestions unless otherwise notedwere in O'Farrell Buffer [OFB]). Digestion was for 16 hours at 37° C.The reaction was stopped by heating to 65° C. for 10 minutes. Theresulting fragments were separated by electrophoresis through a 1%agarose gel containing 0.04M tris-acetate (pH 8.0, 0.002M EDTA) at 40volts for 16 hours. The 1778 bp (1.2 md) Hind III fragment coding forthe influenza HA sequence was isolated from the gel by electroelutiononto Whatman filter paper as described by Girovitz et al., Anal.Biochem. 106, 492-496 (1980).

The Hind III sticky ends of the HA fragment were filled in and Bam HIlinkers added as follows: 500 ng of the Hind III HA fragment in 20 μl1×OFB containing 100 μM of dATP, dGTP, dCTP, dTTP and 6 units of T4 DNApolymerase were incubated at 37° C. for 30 min. The reaction was thenstopped by heating at 65° C. for 10 min. The mixture was then put on iceand 1 μl of 20 mM ATP, 1 μl (1 μg) of Bam HI linkers (pCGGATCCG)(Collaborative Research), 2.5 μl of 2×OFB and 0.5 μl (200 units) of T4DNA ligase were added. Ligation of linkers was for approximately 16hours at 10° C. The ligation reaction was then stopped by heating to 65°C. for 10 minutes and cooled to 37° C. Then, 2 μl (20 units) of Bam HIwere added and the mixture digested for 4 hours at 37° C.

Unligated Bam HI linkers were then removed by spermine precipitation asdescribed in Example XX. Approximately 250 ng of the Bam HI-linked HAfragment were ligated to approximately 180 ng of pRW 120, which had beencleaved with Bam HI and treated with calf intestine alkaline phosphataseto prevent self-ligation, in 10 μl of 1×OFB containing 1 mM ATP and 80units of T4 DNA ligase for 16 hours at 16° C. The entire mixture wasthen used to transform competent E. coli RR1 cells.

10 μl and 100 μl aliquots of the transformed cells were then plated ontoLB agar plates containing chloramphenicol at 30 μg/ml. Cam^(R) colonieswere picked and screened for recombinant plasmids by Bam HI restrictiondigestion of plasmids isolated from minilysates of the transformants.The digests were analysed on 1% agarose gels. Recombinants whichcontained the HA fragment were then screened for orientation of the HAfragment by restriction with Ava I and analysis on 1% agarose gels.

Recombinant plasmids having the HA gene inserted in the Bam HI site ofthe Hind III vaccinia F fragment in opposite orientations weredesignated pDP 122A and pDP 122B.

pDP 122B was used as the donor plasmid for in vivo recombination withVTK⁻ 79 rescuing virus in TK⁻ TS 13 cells to give the new vacciniarecombinant vP 53 (ATCC VR 2060).

EXAMPLE XXVIII Construction of pDP 232B

A DNA fragment containing the coding region for the hepatitis sAg wasisolated from pDP 252 (ATCC 39735) by digesting approximately 15 μg ofpDP 252 with excess Bind III in 1×OFB. The resulting Bind III fragmentswere then separated on an agarose (1%) gel prepared in tris-acetatebuffer. The 1064 bp (0.73 md) Hind III fragment containing the sAg genewas isolated by electroelution into What-man 3 MM Filter Paper.

Approximately 1 μg of this fragment was blunt ended by treating with 4.5units of T4 DNA polymerase for 30 minutes at 37° C. in 10 μl 1×OFBcontaining 2 mM of spermidine, and 100 μM each of dATP, dCTP, dGTP, anddTTP. The reaction was stopped by heating to 65° C. for 10 min. 10 μl of1×OFB containing 2 mM ATP, 2 mM spermidine, 1 μg of Bgl II/PST I linkers(TCTGCAGA, Worthington Biochemicals), and 200 units of T4 DNA ligasewere added to the reaction mixture. Ligation was at 16° C.

After 18 hours, 20 units of Bgl II were added and the reaction wasincubated at 37° C. for 5 hours. The reaction was stopped by heating at65° C. for 10 min. Excess linkers were removed by spermine precipitationof the DNA as previously described (Example XX). The resulting fragment,containing the sAg gene and now having Bgl II sticky ends, was theninserted into the Bam HI site of pRW 120.

Namely, the Bgl II terminated fragment was resuspended in 10 μl of 2×ligation buffer. Approximately 200 ng of pRW 120, digested with Bam HIand treated with calf intestine alkaline phosphatase, was added in 10 μlof H₂ O. The fragments were then ligated with approximately 80 units ofT4 DNA ligase at 16° C. for 18 hours.

This mixture was then used to transform 24 hour old competent E. coliRR1 cells as previously described. 10 μl and 100 μl aliquots of theresulting transformed cells were plated out on LB agar plates containingchloramphenicol at 30 μg/ml.

Several colonies were picked and plasmid DNA from each was isolated byminilysis and analyzed by restriction analysis with Bam HI and Sst I.One such plasmid containing the HBsAg DNA in the correct orientation forexpression from the vaccinia promotor was designated pDP 232B.

pDP 232B was used as the donor plasmid for in vivo recombination withvTK⁻ 79 rescuing virus in TK⁻ TS 13 cells to give the new vacciniarecombinant, vP 59 (ATCC VR 2061).

EXAMPLE XXIX

Construction of pBL 540;

Construction of pBL 310;

Construction of pBL 330 A

CV-1 cells were infected with herpes virus type 1, strain KOS. After24-48 hours, the DNA was extracted from the cells by the techniquedescribed by Pignatti et al., Virology 93, 260-264 (1979). Approximately20 μg of the DNA were digested with Eco RI and the resulting fragmentswere separated on a 1 percent agarose gel run at 35 volts for 65 hours.The Eco RI H-fragment was isolated from the gel by the glass powdertechnique earlier described herein (cf. Example I).

60 ng of pBR 322 were digested with Eco RI and subsequently treated withCIAP in a manner earlier described herein in Example XIV. TheCIAP-treated pBR 322 and about 78 ng of the Eco RI H-fragment were thenligated in 20 μl of 1×OFB containing 1 mM ATP and 40 units of T₄ DNAligase at 10° C. for 16 hours.

The entire ligation reaction mixture was used to transform competent E.coli (RR1 strain) as described earlier herein.

The resulting transformants were grown on ampicillin plates. Thecolonies were then screened for recombinant plasmids by a restrictionanalysis of minilysates as previously described. Restriction analysiswas done with Eco RI to confirm insertion of the Eco RI H-fragment intothe plasmid and with Hind III to determine orientation of the Eco RIH-fragment within the plasmid. The plasmid containing the HSV-1 (KOSstrain) Eco RI H-fragment inserted into pBR 322 were collectivelydesignated pBL 540 (cf. FIG. 19).

One of the plasmids, designated pBL 540A (ATCC 68574), was chosen foramplification in E. coli. After amplification, isolated pBL 540A wasnext digested with Sst I and a 2,900 base pair Sst I fragment known tocontain the entire HSV-1 gD coding region was isolated using the glasspowder method earlier described herein. Approximately 300 ng of thisfragment were blunt ended with T₄ DNA polymerase using the proceduresdescribed in previous Examples.

Finally, 2 g of Bam HI linkers were ligated to the blunt ended fragmentin 20 μl of 1×OFB containing 1 mM ATP, 2 mM of spermidine, and 40 unitsof T₄ DNA ligase at 10° C. for 18 hours. The reaction was stopped byheating at 65° C. for 10 minutes.

15 units of Bam HI were next added and the reaction mixture wasincubated at 37° C. for 18 hours. Residual linker material was removedby precipitation with spermine as described by Hoopes et al. (op. cit.).

All of the Bam HI-linked fragments so obtained were next ligated with300 ng of CIAP-treated pRW 120 (linearized with Bam HI) in 20 μl of1×OFB containing 1 mM ATP and 40 units of T₄ DNA ligase at 10° C. for 18hours.

The entire ligation mixture was used to transform 100 l of competent E.coli (RR1) and the transformed bacteria were grown on agar platescontaining 30 μg/ml of chloramphenicol.

Those transformed E. coli which were chloramphenicol resistant werescreened for recombinant plasmids by restriction analysis of minilysatesas previously described. Restriction analysis was done with Bam HI toconfirm insertion of the Bam HI-linked 2900 base pair fragmentcontaining the HSV-1 gD gene into pRW 120 at the Bam HI site and withPvu II to identify both possible orientations of the fragment within theplasmid. The two new plasmids thus obtained were collectively designatedas pBL 310.

After amplification in E. coli, a Hind III-Bam HI double digest of pBL310 was prepared and a Hind III-Bam HI DNA fragment, approximately 2,500base pairs long and containing the entire HSV-1 gD coding region, wasisolated using the method of Girvitz et al. previously described (op.cit.). Approximately 400 ng of this fragment were blunt ended with T₄DNA polymerase as earlier described. Finally, 2 μg of Bam HI linkerswere ligated to this modified fragment in 20 μl of 1×OFB containing 1 mMATP, 2 mM of spermidine, and 40 units of T₄ DNA ligase at 10° C. for 18hours.

The reaction mixture was precipitated with ethanol at -70° C. for 1hour, re-suspended in 100 μl Bam HI reaction buffer, and digested with15 units of Bam HI at 37° C. for 18 hours. The Bam HI linked fragmentwas isolated from the residual linker material on a 0.6% agarose gelusing the Girvitz et al. Whatman filter paper method.

Approximately 100 ng of this linked fragment were ligated with 400 ng ofpRW 120, previously linearized with Bam HI, in 20 μl of 1×OFB containing1 mM of ATP and 32 units of T₄ DNA ligase at 13° C. for 18 hours.

The entire ligation reaction mixture was used to transform competent E.coli (RR I). The transformants were plated on agar containing 30 μg/mlof chloramphenicol and the chloramphenicol resistant colonies werescreened for recombinant plasmids by colony hybridization as previouslydescribed in Example VI.

Colonies identified as positive by hybridization to a ³² P-radiolabelledHSV-1 gD probe were further screened by restriction analysis ofminilysates with Bam HI to identify insertion of the 2500 base pair BamHI linked fragment into the Bam HI site of pRW 120 and with Pvu II toidentify both possible orientations of the fragment within the plasmid.The two plasmids thus obtained were collectively designated as pBL 330.That plasmid wherein the HSV-1 (KOS) gD insert is oriented with flankingvaccinia virus DNA in the plasmid such as that the direction oftranscription is the same is designated pBL 330 A.

pBL 330 A was used as the donor plasmid for in vivo recombination withvTK⁻ 79 rescuing virus in TK⁻ TS 13 cells to give the new vacciniarecombinant, vP 60 (ATCC VR 2062).

EXAMPLE XXX In vivo Recombination using pDP122B, pDP232B, and PBL330A toGenerate Vaccinia Virus Mutants VP-53, VP-59, and VP-60

50 μg of donor plasmid in 100 μl of H₂ O were mixed in each case with350 μl of 2×Hepes phosphate buffer. Each mixture in turn was mixed with300 μl of vTK⁻ 79 at a concentration of 1.3(10⁶) pfu/ml to which hadbeen added 50 μl of 2.5M CaCl₂.

Each mixture was then added to a monolayer of TK⁻ TS 13 cells [cf. Shenet al., Molecular and Cellular Biology 2 (9), 1145-1154 (1982)] in a 60mm Petri dish to which 700 μl of Eagle's Special medium was then added.At 2 hours after infection, 3 ml of fresh medium were added.

24 hours after infection, the samples were harvested and frozen andthawed three times to rupture the cells.

As described above in Example X, plaques formed on confluent CV-1monolayers under a nutrient agar overlay were then transferred to anitrocellulose filter, a replica filter was prepared, and the originalfilter was subjected to hybridization employing ³² P-labelled fragmentsin each case containing the gene of interest, i.e. HA, HBsAg, or HSVgD.

Positive plaques containing recombinant virus were in each case isolatedfor further purification using the techniques earlier described inExample X.

EXAMPLE XXXI Expression of HBsAg by vP-59 in infected cells

Monolayers of 10⁶ CV-1 cells were infected at 2 pfu per cell with vP 59.After 24 hours, the nutrient medium was collected, the cells were washedwith saline solution, and the wash combined with the supernatant liquid.The washed monolayer of cells was also collected in saline solution.

These fractions were assayed for HBsAg using the "AUSRIA II-125" RIAdiagnostic kit sold by Abbott Laboratories for the detection ofhepatitis B surface antigens. (The test kit employs beads, coated withguinea pig antibody to HBsAg, which are incubated with the material tobe tested. Any sAg present in the sample is bound to the solid phaseantibody. After aspiration of unbound material and washing of the beads,human ¹²⁵ I-sAg antibody is reacted with the antibody-antigen complex onthe bead. The beads are then washed to remove the unbound radioactivematerial. The radioactivity remaining on the beads is counted in a gammascintillation counter, all as described in product literature issued bythe manufacturer in November, 1981.)

In repeated experiments, 150-200 ng of HBsAg were synthesized in a24-hour period using the aforementioned amounts of cells andplaque-forming units. The majority of the antigen was secreted from theinfected cells and was localized in the medium. As shown in the Tablebelow, more than 90 percent of the viral infectivity remainscell-associated so that the presence of the antigen in the medium is notdue to lysis of the infected cells.

                  TABLE                                                           ______________________________________                                                                  % pfu Total   % HBsAg                                    distribu- synthesized distribu-                                            Inoculum Fraction Total pfu tion HBsAg (ng) tion                            ______________________________________                                        vP59   Supernate                                                                               10.9 (10.sup.5)                                                                        7     131     76                                       Cellular 146.0 (10.sup.5) 93 42 24                                         ______________________________________                                    

(For titering the fractions, plaque assays were performed on thesupernatant and cellular fraction. For this purpose, the cellularportion was lysed by freezing and thawing and serial dilutions wereemployed to infect CV-1 cell monolayers.)

EXAMPLE XXXII Expression of HBsAg by vP-11

10⁸ CV-1 cells were infected with vP-11 with 25 pfu/cell. After 24hours, the infected cells were harvested, frozen and thawed three timesto lyse them, and debris was removed by centrifugation for 10 minutes at1,500 rpm. The supernatant liquid was centrifuged for 18 hours at 30,000rpm at 4° C. to pellet the HBsAg. The pellet was resuspended in 1 ml ofPBS and assayed for HBsAg using the commercially available "AUSRIA"assay kit. Uninfected cells were processed in parallel as a control.

The ratio of the positive control mean value to the negative controlmean value was 6.6, indicating the presence of HBsAg in the positivecontrol.

Nys: (FG) rabbits were inoculated intravenously with 1.8 (10⁸) pfu ofvP-11. Serum was collected at weekly intervals after infection andtested for antibody using the "AUSAB" commercial RIA test kit. Theresults are tabulated below.

    __________________________________________________________________________    RIA units/ml serum                                                              Weeks post infection                                                        Rabbit #                                                                           Preimmune                                                                           2 3    4    5    6    7    16   21                                 __________________________________________________________________________    637  0       3.6 × 10.sup.3                                                                5.4 × 10.sup.3                                                               7.2 × 10.sup.3                                                              13.5 × 10.sup.3                                                               7.2 × 10.sup.3                                                               5.4 × 10.sup.3                                                               5.4 × 10.sup.3                636 0 1).sup.3 7.2 × 10.sup.3 13.5 × 10.sup.3 20.8 ×                                                 10.sup.3 18.3 × 10.sup.3                                                18.3 × 10.sup.3 23.5                                                    × 10.sup.3 11.2 ×                                                 10.sup.3                           __________________________________________________________________________

EXAMPLE XXXIII Determination of HSVgD expression by vP-60 rabbits

Nys: (FG) rabbits were inoculated with recombinant virus vP-60 orwild-type virus (VTK⁻ 79) intravenously in an amount of 1.8 (10⁸) pfu.

Serum obtained 3-5 weeks after inoculation was heat-inactivated, mixedwith an equal volume of virus containing 250 HSV (Type 1) pfu as a testdose, and held at 4° C. overnight. The mixture was then plated on CV-1cell monolayers and, after 48 hours, the virus plaques were visualizedby staining with Neutral Red and were counted.

The serum decreased HSV infectivity, as measured by plaque reduction, bymore than 80 percent at a final serum dilution of 1:160 and by 50% at afinal serum dilution of 1:320.

FIG. 20 shows the inoculation, both intradermally and intravenously, ofrabbits with both vP 59 and vP 60. The presence of anti-HBsAg antibodiesin serum collected from these rabbits is shown in FIG. 20. Anti-HSVgDantibodies were shown to be present in serum collected from the samerabbits using standard radiolabelled protein assays on HSV-infected CV-1cells.

Further, the presence of antibodies neutralizing HSV was determined byplaque reduction assays using preimmune serum at a 1:20 dilution as acontrol. The results are shown in the Table below.

    ______________________________________                                        Rabbit #506                                                                                             Reciprocal of                                         Week % reduction serum dilution                                             ______________________________________                                        2           63        80                                                        3 57 80                                                                       4 46 80                                                                       5 74 40                                                                       6 56 40                                                                       7 64 40                                                                     ______________________________________                                    

These experiments established that not only does the production ofantibodies to vaccinia not interfere with the production of HBsAg or thewith production of antibodies to HSVgD, but also that the inoculatedanimal can respond to more than one foreign antigen expressed byrecombinant vaccinia viruses.

EXAMPLE XXXIV Protection of Mice Against Challenge with HSV byImmunization with vP-60

Three sets of Nya:NYLAR mice, an outbred albino strain maintained by theNew York State Department of Health Laboratories, were inoculatedintraperitoneally with either phosphate buffered saline solution,wild-type vaccinia virus (VTK⁻ 79), or with vaccinia virus recombinantvP 60 expressing the HSVgD. Each mouse was injected with 4.5 (10⁷) pfuof the wild-type or recombinant vaccinia virus 0.2 ml ofphosphate-buffered saline.

After three weeks, the mice were challenged with an intraperitonealinoculation of 2.4 (10⁴) pfu per mouse of infectious HSV type 1 (AAstrain).

As is evident from the results shown in the Table below, a vaccine ofthe recombinant vaccinia virus VP 60 conferred protective immunity toHSV.

                  TABLE                                                           ______________________________________                                        Immunizing agent                                                                           No. of mice Survivors                                                                              Survival                                    ______________________________________                                        Phosphate-buffered                                                                         40          18       45                                            saline                                                                        Wild-type 40 12 30                                                            Vaccinia                                                                      Recombinant 40 40 100                                                         vaccinia vP60                                                               ______________________________________                                    

In a further experiment, two sets of mice the same as those mentionedearlier herein were inoculated intraperitoneally with 5 (10⁷) pfu ofwild-type virus (VTK⁻ 79) or recombinant vaccinia virus VP 60.

After six weeks, the mice were challenged with 10⁴ pfu of HSV type 2(Curtis). As is shown in the following Table, the recombinant vacciniavirus coding for the gD glycoprotein of HSV type 1 gave considerableprotection against challenge with heterologous HSV type 2. However, theprotection is not as complete as that observed for the homologous HSVtype 1 challenge.

                  TABLE                                                           ______________________________________                                        Immunogen     # Mice    Survivors                                                                              % Survival                                   ______________________________________                                        Wild type vaccinia                                                                          80        10       12.5                                           Recombinant vaccinia 80 63 78.8                                               vP 60                                                                       ______________________________________                                    

What is claimed is:
 1. A method of replicating DNA in a eukaryotic cellcomprising infecting said cell with a vaccinia virus modified to containsaid DNA, said DNA being exogenous to said vaccinia virus, wherein saidDNA is replicated.
 2. A method as in claim 1 wherein said DNA is alsoexogenous to said cell.
 3. A method as in claim 2 wherein said DNA isexpressed by said cell.
 4. A method as in claim 3 wherein expression ofsaid DNA results in production by said cell of a biological product. 5.A method as in claim 4 wherein said DNA which is replicated includes theherpes simplex TK gene and said biological product is thymidine kinase.6. A method as in claim 4 wherein said DNA which is replicated includesthe influenza hemmagglutinin gene and said biological product is theinfluenza hemagglutinin antigen.
 7. A method as in claim 4 wherein saidDNA which is replicated includes the HBsAg gene and said biologicalproduct is the hepatitis B surface antigen.
 8. A method as in claim 4wherein said DNA which is replicated includes the gene for HSVgD andsaid biological product is the herpes simplex virus glycoprotein D. 9.The method of claim 4 wherein the DNA codes for a herpesvirusglycoprotein and the biological product is a herpesvirus glycoprotein.10. A method for mapping a non-essential region in the vaccinia genomecomprising preparing donor DNA comprising DNA not naturally occurring invaccinia virus present within a segment of vaccinia virus otherwiseco-linear with a portion of the vaccinia genome such that by in vivorecombination the donor DNA can be introduced into a region of thevaccinia genome, introducing said donor DNA into the vaccinia genome byin vivo recombination, recovering recombinants, and determiningstability and viability thereof, whereby viability and stability ofrecombinants indicates that the region into which the donor DNA wasintroduced is non-essential.
 11. The method of claim 10 wherein the DNAnot naturally occurring in vaccinia virus is DNA coding for HSV TK. 12.The method of claim 10 wherein the DNA not naturally occurring invaccinia virus is DNA coding for influenza HA.
 13. The method of claim10 wherein the DNA not naturally occurring in vaccinia virus is DNAcoding for hepatitis B surface antigen.
 14. The method of claim 10wherein the DNA not naturally occurring in vaccinia virus is DNA codingfor a herpesvirus glycoprotein.